Saturday, March 19, 2011

Book Notes: Membranes Part 1

Notes on abbreviations: most abbreviations are mentioned by their full name at least once prior. Any weird abbreviations I used are noted also on the end of each post.
  • organelles are characterized/identified by the proteins on their membrane and in their lumen
    • enclosed by intracellular membranes to separate and specialize different reactions/pathways
    • also need to be able to export products and import necessary proteins
    • nucleus holds DNA, and functions in DNA/RNA synthesis
    • cytoplasm is cytosol plus all the organelles
    • cytosol is the aqueous volume of a cell, site of protein synthesis and degradation
    • ER make soluble and integral membrane proteins and lipids
    • Golgi receives proteins and lipids from ER, modifies them, and ships to correct destination
    • mitochondria/chloroplasts generate energy as ATP
    • lysosomes digest macromolecules and organelles
    • endosomes transport endocytosed stuff to lysosomes
    • peroxisomes sequester dangerous oxidation reactions
  • organelles also characterized by position within cell (e.g. Golgi close to nucleus)
    • organized by cytoskeleton interactions
  • precursor of eukaryotic cells had no internal membranes and performed all membrane functions on the plasma membrane
    • adaptation to internal membranes increased surface area available for all these reactions
    • adaptation to specialization of internal membranes segregated reactions for more efficiency
    • adaptation to enlargement of eukaryotes (much bigger than lil' bacteria) in conjunction with efficiency of energy, storage, transport, etc.
    • most likely evolved from simple folding in (invagination) of membranes and subsequent pinching off into vesicles--this process still occurs in terms of transporting goods from one organelle to the next
    • mitochondria and plastids evolved from endosymbiosis (evidenced by their own separate DNA)
  • sorting signal: part of A.A. sequence of polypeptide of protein, directs delivery of newly synthesized protein to location out of cytosol (into an organelle for example)
    • proteins without sorting signals remain in cytosol permanently
  • 3 methods of protein transport:
    • gated transport: protein channel that only allows passage of transporters, molecules need transporters to move them through channel (ex. NPC of nucleus)
      • used for transport between spaces that are similar (nucleoplasm and cytoplasm)
    • transmembrane transport: protein channel that allows passage of molecules with correct signal, molecules that are supposed to go through unfold to go through (ex. movement into ER or mito)
      • used for transport between spaces that are not similar (ER lumen and cytoplasm)
    • vesicular transport: comparatively less selective, membrane buds off and takes whatever is in its lumen into another compartment, inside goods never see what's outside the membrane
      • used for transport between spaces that are similar but requires going through space that is not similar (from ER lumen to Golgi lumen but through cytoplasm)
  • signal sequence: A.A. sequence at the N-terminus that contains the sorting signal
    • signal peptidase cleaves the sequence once the protein has been delivered to the destination
    • signal patch: a type of permament signal sequence in the middle of a polypeptide that forms a 3D area when the protein is folded and is not cleaved (e.g. NLS or NES for nuclear transport)
    • ER signal is usually 5-10 hydrophobic residues at the N-terminus
    • not continuing to Golgi involves a specific sequence of 4 residues at the C-terminus
    • mitochondria signal is alternating positive and hydrophobic residues
  • sequences are identified by using site-directed mutagenesis and see where proteins end up after you mutate a part of its sequence (if you mutate a section of the sorting signal, the protein goes somewhere else, and then you sequence that important part to identify the sorting signal)
    • same destinations may have different sequences: characteristics like hydrophobicity are more important than the actual A.A.
    • similarly, receptors recognize classes of sequence signals rather than be specific
  • 3 methods of studying protein translocation:
    • transfection: fuse cytosolic protein with a signal sequence, transfect cell with the cDNA of this fusion protein, let the cell express it, determine where the protein ends up by immunostaining or cell fractionation.  THEN, do site-directed mutagenesis to see which residues of the sequence are most important.
    • biochemistry: perform in vitro translation of a protein that has the signal of interest (SgOI), label with radioactive A.A., place in proximity of isolated organelle, and see if it translocates
      • it translocates into the organelle if:
        • 1. labeled protein cofractionates with organelle in centrifugation
        • 2. isolated protein moves faster than control protein through gel because the organelle cleaved the signal
        • 3. can still isolate intact protein when you add nucleases because the organelle is protecting it, but detergents that remove the membrane allows nuclease digestion)
    • genetics: engineer mutations in the translocation machinery, wait to see if cell dies because an important protein went somewhere else or couldn't go to destination at all
  • most organelles cannot be made de novo
    • some can be produced from budding off of others
    • neither can specific translocation machinery be produced from scratch

pg. 695-704 (from Chapter 12)
A.A = amino acid

Book Notes: Apoptosis

I take notes from the book Molecular Biology of the Cell, written by Bruce Alberts, Alexander Johnson, Julian Lewis, etc, etc.  published by Garland Science, Taylor and Francis Group, LLC, copyright 2008.  Obviously some phrases or sentences are written verbatim from the book because these are notes, not intended to be any form of plagiarism.  Page numbers will be listed at the very end of every post.
  • programmed cell death: controlled manner of suicide initiated from inside cell
    • apoptosis: one form of programmed cell death, identifiable by changes in morphology (organelles collapse, chromatin fragments, cell blebs into mini vesicles called apoptotic bodies, which get engulfed by macrophages)
    • no inflammatory response to cell loss
  • necrosis: cell death resulting in swelling and bursting, very messy!  initiated by external injury
    • mess induces inflammatory response
  • apoptosis clears unwanted cells (e.g. eliminate webbing between fingers)
    • also removes damaged/mutated/infected cells
    • must be tightly regulated with cell division to control cell count in tissue
      • e.g. cut out piece of liver, division increases to replace cells; enlarge liver, apoptosis increases to remove extra cells
      • both result essentially in going back to normal shape/size liver
  • apoptotic cells are identifiable by biochemical changes
    • DNA is cleaved into fragments of specific sizes, banding pattern of fragments from gel electrophoresis is specific to apoptotic cell
    • Tdt enzyme adds nucleotides to the 3' end of fragmented DNA, engineer labeled nucleotides to visualize local apoptosis in tissue: TUNEL method
    • phosphatidylserine: phospholipid exclusive to inner membrane leaflet, flips to outer membrane during apoptosis, can be visualized by labeled Annexin V protein that binds to it
      • lipid acts as a "eat me" signal to induce phagocytosis by neighbor macrophages and to block inflammation response
      • apoptotic cells also lose the "don't eat me" signal (was observed that macrophages ate anything and everything including beads without any signal, so healthy cells must have a "don't eat me" signal
    • loss of membrane potential = loss of active formation of electrical gradient, so charged dyes naturally diffuse into certain organelles to neutralize charge, visualize apoptotic cells by the presence of the dyes in organelles
    • cytochrome c released into cytosol during apoptosis, observe by any method of localizing proteins (i.e. immunolocalization)
  • apoptosis triggered by proteolytic cascade (as opposed to phosphorylation cascade)
    • caspase: protein with Cys at active site and cleave at Asp on target proteins, synthesized as inactive precursor procaspase, which must be cleaved to be activated
      • initiator procaspase: when activated, goes to cleave other procaspases
      • executioner procaspase: when activated, goes to cleave other procaspases and other target proteins (e.g. lamins, endonucleases, cytoskeleton, cell-cell adhesion proteins)
      • caspase cascade is IRREVERSIBLE
    • different cell types have different types of caspases
  • cells continuously make apoptosis proteins whose activation depends on a signal (as opposed to cell division, when protein transcription depends on a signal)
    • caspase recruitment domain (CARD): within "prodomain" of initiator procaspase, recruit each other and assemble with adaptor proteins into "activation complex"
    • cleave each other in the assembly and are all activated (this is the irreversible point)
    • two pathways: extrinsic vs. intrinsic
  • death receptor: surface transmembrane protein that binds ligands outside of cell, and has "death domain" inside cell
    • homotrimer, belongs to TNF receptor family (tumor necrosis factor) of which includes the Fas death receptor
    • ligand is also a homotrimer belonging to the TNF signal family
  • extrinsic pathway begins with cytotoxic "killer" lymphocyte coming over to tell a cell "it's time to die"
    • lymphocyte has Fas ligand on its surface, binds to Fas receptor on victim cell, death domains recruit adaptor proteins that recruit initiator procaspases
    • procaspase-8 and/or procaspase-10 form death-inducing signaling complex
    • procaspases activate themselves in the DISC, cell begins to die
    • sometimes extrinsic pathway recruits intrinsic pathway to amplify death-dealing cascade
  • exist inhibitory proteins that act to restrain the extrinsic pathway
    • decoy receptors: bind ligand but have no death domain, competition for ligand binding inhibits real receptors
    • FLIP: fake initiator procaspase without proteolytic domain, competition for DISC binding sites inhibits real procaspases
    • both prevent false/inappropriate apoptosis activation (boy who cried wolf, except irreversible)
  • death receptors also activate other pathways, depends on what signal they receive
    • e.g. TNF receptors activate NFkappaB pathway promotes cell survival and inflammatory response
  • intrinsic pathway occurs when cell is stressed (such as by midterms)
    • cytochrome c: component of ETC (remember making ATP?), released from mitochondria to cytosol
    • Apaf1: activated by cyt c binding, multiple Apaf1 bind to each other at CARD domain to form heptamer wheel called apoptosome
    • procaspase-09 come together on the apoptosome and activate each other like on DISC
  • Bcl2: protein family that regulates the intrinsic pathway, control release of cyt c and other proteins into cytosol
    • some promote (pro-apoptotic) and some inhibit (anti-apoptotic)
    • two opposites can bind to each other and inhibit each other
    • Bcl2 proteins share BH domains (Bcl2 homology) 1 through 4
  • pro-apoptotic proteins are BH123 (lack domain 4) and BH3-only (only homologous at domain 3) proteins
    • apoptosis signal triggers BH123 proteins to activate and aggregate on mitochondrial membrane, inducing cyt c release
    • Bak and Bax are the main players that heterodimerize, where Bak is always found on membrane but Bax must be moved from cytosol to mitochondria membrane in response to signal
      • activated by BH3-only proteins
      • also function on ER to release calcium ions
  • anti-apoptotic proteins are Bcl2 and Bcl-XL, also located on ER and outer mito membrane, and on nuclear envelope
    • preserve membrane integrity by binding to pro-apoptotic proteins and inhibiting their dimerization (poor Bak)
    • inhibited by BH3-only proteins to induce apoptosis
  • BH3-only proteins promote Bak and Bax aggregation and inhibit anti-apoptotic proteins
    • communicates signals to apoptotic pathway (e.g. DNA damage -> p53 -> expression of BH3-only proteins Puma and Noxa -> intrinsic apoptosis)
    • is link btwn extrinsic and intrinsic pathway: caspase-8 cleaves BH3-only Bid into tBid, who visits mito and inhibits anti-apoptotic Bcl2's and triggers Bak and Bax aggregation
    • Bid, Bim, and Puma act on all anti Bcl2's, where others are much more specific
  • inhibitors of apoptosis (IAP): proteins that inhibit apoptosis (I never knew!)
    • have 1+ BIR domains (baculovirus IAP repeat): enable binding+inhibiting of activated caspases, some polyubiquinate caspases
    • threshold of inhibition that must be overcome in order to have apoptosis
  • anti-IAP: proteins that bind to IAP's BIR domains (takes up the active site)
    • examples include Reaper, Grim, and Hid
    • held in mitochondrial intermembrane space, released when apoptosis signal received
  • survival factors: extracellular molecules that signal inhibition of apoptosis
    • some cells require continuous supply of survival factors (cell lives only if organism needs it)
    • e.g. nerve cell gets survival factors from the cell it connects to (if it doesn't connect anywhere, it is useless=so it dies through the lack of survival factors)
    • bind to cell-surface receptors that may increase anti-apoptotic Bcl2 production or inhibit BH3-only proteins or activating IAP and deactivating anti-IAPs
    • lack of survival factors causes increase in pro-apoptotic BH3-only proteins
  • autophagy: cell eating itself, caused by lack of food import into cell
    • when genes for pro-apoptotic proteins have been knocked out, so cell doesn't do apoptosis when there is a lack of survival factors
    • but no survival factors means inefficient or no food import, which leads to starvation/munching on yourself by inserting organelles/cytoplasm into lysosomes
  • too much and too little apoptosis can cause disease
    • excessive apoptosis = tissue damage (e.g. something like heart attack kills off some cells, but then lack of blood flow leads to other cells committing suicide)
    • confused cells, who don't die when they're supposed to, start accumulating in places, and then immune system goes to attack them (why won't you guys die?!) = autoimmune disease
    • decreased apoptosis also contributes to cancer (i.e. mutated p53 can't activate apoptosis despite increased mutation or DNA damage, leads to uncontrolled mutated growth)
pg. 1115-1129 (i.e. all of Chapter 18)

Tuesday, March 15, 2011

Test Commentary

So, there are some things to complain about this round of testing:

(1) It was rather like a jigsaw puzzle than a knowledge test (regulator of an inhibitor and inhibition of said inhibitor)
(2) There were things on there that didn't exist in the book (H3K9)
            ---The only reason I lucked out was because I happened to be looking at histone acetylation on Wikipedia while studying, and Wiki mentioned the H3K9 and H3K27 as referring to the respective lysines).
(3) I'm sure there are multitudes of things that confused you.  If you have suggestions on how I should present material differently, please comment or message me.  What I'm going to do is to put all textbook notes I take on here AND any extraneous information I happen to find while on Wikipedia/Google.  I will still synthesize my study guide based on the lecture summaries, though, b/c that's really the meat of things.

P.S. I'm very sorry to the people who don't approve of my review session/study guide.  No one is being forced to go, and if it isn't helping people, why would they attend?  As long as someone wants help, I'm very happy to help them.  If everyone can get A's and B's, why not?  I have heard that the intention of making CellBio extremely hard is to weed out the premeds who aren't really interested in science/med, but even non-premeds with certain majors HAVE to take this class.  I think the idea should be to present Cell Bio in an interesting (if possible) and learnable way (Dr. Klein, who by the way is an awesome Orgo Chem prof, shares similar views), and people who really don't like science or medicine will fall out anyway with the MCAT (which is already crazy difficult), but that doesn't mean the class should not be passable (I have heard that a large chunk of the class last year had D's or lower).  Anyway, that's just my point of view.

Sunday, March 13, 2011

Study Guide for Cell Biology: Nucleus, Cell Cycle, Mitosis

Nuclear Transport

I enjoyed this skit.  We might have a whole cell broadway show someday.  ;)
I was tricked by the practice problem.  Ran BP (which is not related to NTF2) is an importin Beta, but I'm not really sure what it does.  It wasn't mentioned in lecture summaries or the book...

Cell Cycle. What can I say?

For clarification, most of the lines have either an arrowhead or yellow cross in place of an arrowhead.  Lines that do not have either are binding to the molecule whose action line they intersect.  A yellow cross as an endpoint indicates the source of that line is inhibiting the action of the line they cross (i.e. there is a red line that ends in a yellow cross from G1-Cdk that crosses Rb's action line=G1-Cdk inhibits Rb).