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Molecular Stress > week 9-end > Flashcards

week 9-end Flashcards

1
Q

what is osmosis, what is osmotic pressure

A

spontaneous diffusion of water molecules across a semi-permeable membrane towards side with more solutes

osmotic pressure determined by Van’t Hoff Eqt (pi = RT (Cb-Ca)
- ions or non-ions count
- 1 M of glucose is equal to 0.5 of NaCl (because it separates in water into 0.5Na + 0.5Cl)

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2
Q

what’re the effects of Osmosis on cell sizes, what is the rate that it happens at, what is an example of this happening

A

cell membranes are semipermeable, and amt of water going across the plasma membrane determines cell volume (swell/shrink)

  1. isotonic/isosmotic = not shrinking/swelling (150nM NaCl)
  2. hypertonic solutions makes cells shrink
  3. hypotonic makes cells swell

rate: happens fast

ex. erythrocytes are normally biconcave disks but will shrink or swell depending on solute concentration outside

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3
Q

what are aquaporins, explain the experiment done w/frog oocytes, and the structure of aquaporins,

how many aqua porin families are in humans, what’s an important one

A

water-channel proteins that allow biological membranes to be permeability compared to regular lipid bilayers

experiment
- frogs are impermeable to water and will remain unchanged in hypnotic solutions but when injected with aquaporin mrnas they began to swell (showed aquaproins specifically increase bio membrane water permeability)

functional form:
tetramer of identical 28kDa subunits embedded in the membrane that form a channel each

  • a single aquaporin has 6 transmembrane alpha helices connected by 2 hydrophobic loops that form the Chanel pore
  • inside, water makes hydrogen bonds with channnel-lining hydrophilic amino acids (rapid water diffusion while blocking ions)

humans: have 11 genes
- aquaporin 2 inactivation causes diabetes insipidus (kidney disease where large amts of dilute urine is excreted cause kidneys can’t reabsorb water)

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4
Q

what is the osmolality of body fluids and tissues in animals, how is it calculated

A

hyperosmotic conditions in animal tissues are not unusual
- lymphoid/hepatic tissue is hyperosmolar compared to blood/brain/lung

osmotic conc. is measured by osmoles (total of solute particles both molecules and ions)

osmolality (mOsm) of a solution is number of osmoles/kg of solvent

osmolality varys from freshwater species (225) to marine species (1000)
- we are at 300mOs/kg and our kidneys are at 1500 showing that animal cells have systems that allow adaptation to hypertonic conditions

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5
Q

what is osmotic stress, what is our cellular response to it, what is the most common inducer of osmotic stress

A
  • shock happens when solute conc. around cell suddenly changes
  • response is rapid (seconds) and changes intracellular conc. of ions such Asas K+, Na+, and Cl-
    1. change in cell volume is corrected by RVI and RVD mechanisms
    2. cytoskeletal reorganization
    3. activation of integrins, RTKs, channels
    4. **all done through activation of specific transcription factors (TonEBP) and related signal transduction networks leading to production of organic osmolytes

most common inducer of stress: hypertonic conditions

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6
Q

how does hyperosmolarity induce the actin cytoskeleton reorganization

A
  • experiment where hamster ovary cells (CHO) in isotonic medium was a treated with 300mM sucrose for 10 mins (hyper osmotic condition cause sucrose is impermeable)

this showed
- untreated CHO cells under iso conditions have well-organized cytoskeletons when visualized under f-actin stain

hypertonicity induces peripheral accumulation of f-actin and reduces stress fibres in the cytoplasm to helps cells resist increased osmotic pressures

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7
Q

explain the TonEBP pathway in animal cells

A
  1. all is controlled by activating the TF TonEBP through its nuclear redistribution, dimerization, and phosphorylation
  2. TonEBP binds to TonE enhancer elements in the nucleus and induces synthesis of multiple osmoprotective genes that allow renal cells to adapt to high osmolality through accumulation of non-ionic organic osmolytes/compatible solutes (no death)
    (SMIT= inositol
    AR = sorbitol
    BGT1 = betaine)
  3. OR it could stimulate synthesis of HSP70, AQP2, UTA to target certain aspects of osmotic stress

summary (TonEBP activation either makes compatible osmolyte that maintain water homeostasis or others to take care of some things)

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8
Q

what are the functional domains of TonEBP and NFAT5

A

TonEBp or NFAT5 are members of: mammalian rel-like TFs containing RHD needed for DNA binding

  • NFATS are TFs that regulate (t-cell proliferation/immunity) and other developmental roles,
  • NFAT5 specifically lacks Ca2 docking sites and its domains are sensitive to hypertonia, allowing it to activate TonEBP in response to hypnotic situations

7 domains:
TAD 1 and 2
RHD
DD
and nuclear transports NES, AED, and NLS

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9
Q

explain the hypertonicity signalling to TONEBP

A
  1. post-translational modification of tonEBP (phosphorylation to facilitate rapid nuclear localization and then possibly summoylation)
  2. interactions w/RHA, PLC, and ROS
    - binding of RHA inhibits its binding to RHD, allowing TonBP to be dimerized
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10
Q

what is the bidirectional regulation of TonEBP activation

A

TonEBP activity/nuclear localization decreases when ambient tonicity is lowered

increased when ambient tonicity is increased

nuclear localization of TonEBP is controlled by inactivation of NLS as site-directed mutagenesis prevents tonicity effects

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11
Q

describe the structure of the TonEBP-DNA complex

A
  • its dimerization domain is part of DNA-binding domain RHD
  • it works by forming a complete circle around DNA
  • one monomer binds to TonE TGGAAA seq while the other binds to the nonconsenus seq through backbone contact
  • thus, TonEBP binds its TonE increases kinetic stability of the TonEBP–DNA complex.
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12
Q

what is the dual modes of TonEBP activation

A
  1. nuclear redistribution of available TonEBP for immediate response
    - (nuclear localization determined fast by ambient toxicity due to sensitive NLS domains)
  2. increase in abundance of TonEBP mRNA for long term adaption to stress
    - (NFAT5 is late-response gene and is the slowest amongst stress response)
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13
Q

what is the ToneEBP expression in tissues

A
  • is ubiquitously expressed at both mRNA and protein levels for most tissue
  • its role in these tissues are explanatory asa kidneys and such are exposed to hypertonic conditions
  • its expression in other tissues not normally in hypertonic conditions show that it may be a general safety system protecting against pathological hypertonicity (eg. the brain)
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14
Q

What are essential metals, where do they occur, what is the most prevalent ones.

A

they are cofactors required for catalytic activity of 4-% of enzymes (metalloenzymes)

  • metaloenzymes occur in all 6 enzyme commission classes (oxidoreductases, transferase, hydrolase, lyases, isomerases, and ligases)

prevalent:
- Mg
- Zn
- Fe
- Mn

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15
Q

what is the toxicity of metals, what does it do to cell, how is this caused

A

WHIMIS 2015 states that:
As, Be, Cd, Cr, Co, Pb, Hg, Ni, V are all toxic when exposed even at low levels (health hazard/acute toxicity)

damages: cellular damage, inflammation, cancers, immunity/kidney/respiratory impairment, and neurotoxicity

Cause: interaction with SH+functional groups displacing essential metal cofactors and binding w/certain proteins
(su, fe, se, zn are essential but longterm is toxic)

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16
Q

what is the zinc-finger motif?
what is it’s most common composition?
what % of the proteome does it make up?

A

type of DNA binding domain where metal stabilizes proteins (3 secondary structures held together by zinc ion)

composition:
- 2 antiparallel B-strands + 2 Cysteines + (1 A-helix + 2 histines)
- these coordinate the zinc ion forming stabilized finger shape

rare secondary type: C4 zn-fingers that conserve 4 cysteines in contact with Zn ions

3%

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17
Q

How does the zinc-fingers interact with DNA

A
  • zinc fingers wrap around the DNA double helixes by going inside the major grooves (recognition between finger and its target)

many TFs contain multiple C2H2 zinc fingers inside them

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18
Q

what are the mechanisms of MTF-1 regulation

A
  1. loading all F1-F6 in the MTF-1 w/zinc (directly or indirectly through zinc release by metallothioneins who oxidatively stress/comptitive heavy metal load),
  2. dimerization, and phosphorylation (must happen)
  3. MTF-1 interacts with Crm1 or exporting 1 to shuttles from cytoplasmic to nucleus (cause it has a NES)
  4. binds to MRE of responsive genes and cooperates with SP1 and P300 to drive gene expression (maintaining metal homeostasis)
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19
Q

explain the metal stress response pathway, explain the functional domains of the TF for this pathway

A
  1. metals activate MTF-1which binds to the short dna-seq motif (MRE) in promoters/enhancers of genes encoding metallothioneins and metal transporters

MREs are similar between human and drosophila
- both MTF and MRE are conserved

MTF-1 is a Zn-finger, domains:
- in the nucleus end, nuclear import signal (NIS)
- DNA binding domains: F1-F6 (Zn-fingers)
- unclear export signal (NES) right after
- transactivation domains (acidic, pro, and Ser/Thr)
- homedimerization domain end

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20
Q

who are the main interaction partners of MTF-1

A

HIF-1a (helps MTF1 activate MT-1 for hypoxia response)

NrF1 (helps activate basal mt1/2 in mouse liver)

HSF1 (reduces metal-induced dependent Hsp70 expression)

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21
Q

what’re the subcellular distribution of MTF-1

A
  1. human embryonal kidney (HEK293) cells were transfected with VSV-tagged MTF-1 and fluorescent immunostaining (used to visualize MTF-1 after different treatment)
  • VSV is an 11mer virus G protein used as a tag for transfected protein that can be detected w/antibodies
  1. results show untreated cells have MTF-1 localization in the cytoplasm

cells treated with zinc/cadmium showed MTF-1 moving to nucleus

metal stress response can be stimulated by LMB, a drug inhibiting NES binding site of the CRM (exporting)

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22
Q

what is the nuclear import of MTF-1 that is induced by other stress stimuli

A
  1. by heat shock (43ºC, 1hr)
  2. hydrogen µM, 3hrs)
  3. low pH (6, 1hr)
  4. serum (10% dialyzed FBS, 3hrs)
  5. cyclohgeximide (10µg/ml, 3hrs)
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23
Q

what are mtallothionneins

A

cysteine-rich, 7kDA weight, intracellular metal binding proteins required for metal homeostasis / detoxification

MTF-1 induces genes that encode for them, there are at least 11 different genes encoding for them

24
Q

Break down the domain structure of metallothioneins

A

a-domain and b-domain that can bind 3-4 atoms of metal ions respectively

  • the domains have different binding affinities to metals
    Zn2+ < Cd2+ < Cu2+ < Ag+ = Hg2+ = Bi3+
25
Q

what is genotoxic stress and what’re the consequences of DNA damage

A

insult or deleterious effect of exogenous (environmental) or endogenous (chemical/physical) damages on genes and related functions
- endo happens frequently and include altered bases
- exo associated with bulky lesions and breaks

consequences:
- changes to genome = errors in transcription of DNA = Bad proteins

  • cells that can’t repair damage will
    1) cells become senescent (irreversible dormant)
    2) apoptotic
    3)malignant (cancer
  • can be carried over to daughter generations if not repaired
26
Q

What are the DNA responses in genotoxic stress

A

a complex signal transduction pathway using enzymatic tools/mechanisms:
- base excision
- mismatch excision
- nucleotide excision repair mechanisms
- DNA repair alkyltransferases
-homologous recombination
- non-homologous end-joining (few redundancy)

27
Q

what’re some potential sites of DNA lesions

A

Sites on the DNA that affect point mutations/breaks

  • chemically sensitive amino side-chains of nucleotide based (ACTG)
  • phosphoester bond in backbone
  • N-Glycosyl bonds linking pentoses
28
Q

what’re the damages from cellular environment (genotoxic stress)

A
  1. depurination:
    • spontaneous hydrolysis of N-glycosyl bond (usually a purine) resulting in AP site
  2. Deamination
    • spontaneous removal of AA from ACG making them Uracil, xanthine, and hypoxanthine
  3. oxidative damage (ROS) causing CG->AT substitution
29
Q

explain the steps of Base excision repair, when is it used

A
  1. Specific DNA glycosylase recognizes TG mismatch and damaged base, flips base outwards from double-helix and hydrolyzes the N-glycosidic bond leaving AP site
  2. APE1 cuts dna backbone at AP site
  3. AP lyase excises the deoxyribose-phosphate-AP site from DNA leaving a gap
  4. DNA polymerase B fills gap, Ligase seals

used:
prior to replication in CT mismatch caused by deamination

30
Q

explain the steps of mismatch excision repair?
when is it used?
what is one disease that prevents it from working?

A
  1. MSH2/MSH6 protein complex recognize mispaired segment
  2. ENDONUCLEASE complex (MLH1/PMS2) cuts synthesized daughter strand , HELICASE unwinds helix, and EXONUCLEASE removes nucleotides
  3. DNA polymerase fills and ligase connects new section to backbone

used:
after replication for mismatches and in/dels made by DNA polymerase

loss-of-function mutations in MSH2/MLH1 causes Lynch syndrome or non-polyposis colorectal cancer = mutant proteins normally needed for this pathway

31
Q

what is DNA damage caused by alkylation of bases, what is cross-link alkylation

how is it fixed

A

alkylating agents induces H-bonds in the double helix (innocuous, N7 is harmless, N3 causes cytotoxic noncoding base, O6 by EMS transferring ethyl causes mutagenic)

some agents have 2 reactive sites creating interstrand crosslinks. Chemical warfare (mustard gas) but derivatives are now used for cancer treatment cause of cytotoxicity

06methylquaninmthytransferase (suicide enzyme) irreversibly inactive by alkyltransferase single-step alkyl DNA transfer to cysteine residue in centre to restore G

31
Q

what is DNA damage caused by UV radiation (exogenous) and how is it fixed, what’re exceptions, what is a disease that makes this damage more susceptible

A

sun (260nm) causing formation of thymine-thymine dimers making DNA kinks

corrected by global genome NER
1. XP-C23B sees pyramiding dimer kinks and recruits TFIIH to unwind helix using atp-helicase, XP-G and RTPA to exasperate this and form 25base bubble
2. XP-F and XP-G now cuts damage +- 24-32 bases and lets it degrade then dnapolymerase and ligase replicate and seal.

exception transcription-coupled NER
1. rna polymerases sees lesion instead and recruits CSB triggering helix opening, recruits TFIIH etc.
xeroderma pigmentosum= 1000x+ more likely to get skin cancer

32
Q

what is damage caused by ionizing radiation and how is it repaired, what’s its relation to hereditary breast cancer

A

exogenous energetic gamma/x-ray ionizes h20/radicals around dna that attack bass and creates breaks

dsDNA breaks are fixed by NHEJ(ligation of breaks, template independent, at any phase of cycle, limited loss unless multiple breaks whose mistake can be deleterious)
1. KU70/80+DNA-PK binds to breaks and forms synapses between ends
2. KU+DNA-PK recruits and activates proteins+endonucleases to trim hangers and ligase joins ends

or
HR (accurate using template to repair, only in S or G2)
1. PARP and MRN sensor complex recognizes dsB and mediate initial resection
2. ATM activates proteins+BRCA1 form overhang 3’ ssDNA ends coated w/RPA
3. BRCA2 displaces RPA with RAD51 to activate overhang and catalyze invasion of 3’end into sister chromatid
4. Holliday junctions form and separate.

BRCA1/BRCA2 mutant allele (reg 11% but BRCA1=65%. BRCA2=45% chance)

33
Q

list all the different guest lecture contents

A

Patrick lajoie: TUDCA alters internalization of anti fungal drugs: differentially controls anti fungal drug bioavailability to reg efficiency and can act independently if its role has a chemical chaperone. Must be present with antifungals to exert facts on cell growth and only rescue pharmacologically-induced er stress. IRE1 required for anti fungal resistance and LBS-activated UPR is transcriptionally different

Martin Duennwald: genetics, environment and aging (increas ROS, shortened telomeres, cycle arrest, calorie restriction delays aging, reduces aggregation) affects misfolding in neurodegenerative disease . DnaJC7 reduces TDP-43 and FUS toxicity and modulate its subcellular localization. OxPhos exacerbates Asyn Toxicity

34
Q

what’re the cells and mediators of the inflammatory response

A

Innate immunity (immediate/non-specific) in tissues (macrophages, dendritic cells) and circulation (leukocytes) detect PAMPs and DAMPs(damages) w/ Toll-like receptors(TLR) arrow from here for next slide point

dendritic cells recognize pathogens and release inflammatory cytokines/chemokins
neutrophils and machrophass kill pathogens and accumulation of leukocytes=pus

35
Q

cellular components of innate immune system

A

majority of innate immune cells have cell-surface receptors(TLR) activated by PAMPS

Inflammatory markers:
TNF-alpha, IL-1B and IL-6

signalling pathways are functioning in inflammatory response, NF-KB is ssntial in conjunction w/most cytokins

36
Q

TLR, structure?

A

evolutionary ancient pathogen recognition/signalling system discovered by Nusslein-Volhard. mutant toll flies susceptible to fungal infection showed it immunity involvement. there’s 10 in humans, 12 in mice.
structure phosphorylates/signals downstream = release of NF-KB and

transcription of inflammatory genes:

extracellular (leucine-rich sickle-shaped repeats involved in ligand recognition)

transmembrane:
cytoplasmic: subdomains TIRs recruits 6 adapter proteins (MyD88 w/DD domain that recruit IRAK4/2 that also hav dd domains)

specificity and local (all functions as dimers): pathogen-derived nucleic acid (TLR3,7,8,9,13) @ intracellular membrane of endoscopes where they detecting DNA/RNA of degraded pathogens and unmethylated CgP dinulceotide

and
patho-derivid-cell wall component (TLR1,2,6,4,5,11,12, proteins) sensors @ extracellular space that recognizes lipoteichoic acids of gram-positive bacterial wall, LPS of outer membrane , other fungal cell-wall of glycans/proteims.

37
Q

what is the NF-KB stress response pathway

A

TLRS trigger this pathway. It is a transcriptional driver of inflammatory response genes like cytokines,chemokines,COX-2,Cytochrome P450,HSP90, addhesion proteins (binding sites are in promoter region) common drug that inhibits COX is ibuprofen.

families
1. NF-KB/rel (rela/p65, relb, c-rel, p50/p150, p52/p100 all sharing RHD that can form dimers that regulate gene expression) by binding to kb sites
2. IKB (8 proteins, p105 and p100 are non-processed)
3.IKK (catalytically active kinases IKK1, IKK2 and regulatory scaffolding protein, NEMO (NFKB essential modifier))

canonical NF-KB components:
1. heterodimer TF RelAA(P65)/p50 (NF-KB1)
2. IKBa sensor that inhibits NF-KB
3. trimeric IKK complex transducer(alpha/beta subunits + regulatory)
pathway (slow):
1. toll-like receptors trigger trimeric IKK by phosphorylation of IKKB subunit induced by TAK1
2. beta subunits phosphorylates inhibitor I-KBalpha which bind E3 ubiquitous ligase which induces polyubiquination of it
3. degradation of I-KBA unmasks nuclear-localization signals in both subunits of NF-KB = translocates to nucleus
4. transcribes numerous inflammatory genes to terminate signalling (neg feedback)

non canonical NF-KB components and steps
1. TF (relB/p52, NFKB2)
2. p100 sensor
3. IKK alpha kinaas transducer with only 1 catalytic subunit
*associated w/lymphoid malignancies, b-cell survival, bone metabolism, and dendritic cell activation
pathway (rapid):
1. relies on phosphorylation-induced p100 processing into p52 triggered by TNFR signalling (needs NIK kinase and IKKalpha but not trimeric IKK and TAK1)

38
Q

what is glycocalyx

A

carbohydrate rich zone on cell surface. visualized by ruthenium red stain and affinity for lectins (dark thick layer around cell in stains). made up of glycan chains (glycoproteins+glycolipids+adsorbed glycomoleculs). Protects cells from mechanical/chemical damage and social distances from foreign objects. can be involved in transmembrane signalling and intercellular comms.

in diagram: lipid bilayer, chunks ontop is major proteins, complex branches is carbs(glycocalyx), 10nm red blood cells, 200cell lining

made of: 9 monosaccharides (hexoses, 6C’s). cn be linear or ring forms

D-Glucose has interconvertible forms :
Linear(open), pyranose ring, furanose ring

39
Q

what’re glycosidic linkages

A

formed between anomeric C of one monosaccharide and a hydroxyl of another. Makes glycans special cause they canna form diff amts of glycosidic bonds

N-linked glycans (GlcNAc in invariably B-linnked to the amide nitrogen of an Asp and first sugar attached)

O-linked are attached to OH of Sir or Threonine and has disaccharide core, size ranges up to 1k-monnosaccharides

typical branched “biantennary” N-glycans
chem build of N-Linked with common pentasachhaaride core but diff terminal extensions cause branches.

40
Q

Classes of animal glycans and glycoconjugates

A
  1. Glycoproteins (glycosylated proteins covakebtly linked by N/O-glycans assembled in ER-Golgi path
  2. GPI (glycol bridge b/w lipid anchor and c-term)
  3. EGF/TSR (mem proteins glycosylated w/short unbranched carbs)
  4. Proteoglycans (glycoconjugates w/1+ attached to core protein, linear polysaccharides)
  5. Glycolipids (glycol attached via glucose/gaalactose to OH group of lipid)
41
Q

Lectins

A

carb/glycaan binding protein w/no enzymatic activity to sugars. ubiquitous and sugar specific. Has min 1 CRD responsible for binding glycans. if added to cell suspension, will bind to surface glycans, form bridges, induces cell aggregation.

animal lectins:
1. galectins (soluble galactose binding)
2. c-type (transmit w/Ca depnd. glycan specificity.
3. p-type (transmeme spec. to mannose6-phosphate)
4. I-type (transmem w/ immunoglobulin domain)

human galectins structure (his research)
- recognized b-galactoside containing glycans. similar to CRDs and soluble/multifunctional
- in/outside cell whose CRDs ate beta-sandwiches w/2sheets of B-strands on concave(S1-S6) and 5B-strands on convex side (F1-5)
- types: (proto-type w/1 CRD, Tandem-repeat w/2diff CRDs, chimeric w/1CRD linked to non-lectin N-term)

tissue specific:
LGALS12: highest RNA expression in adipose tissue, breast, and bone marrow.also a marker for diff phenotypes of neutrophils
LGALS16: highest in normal placenta, retina, and brain tissues

42
Q

categories of galactic genes in cellular stress response

A

stress-inducible
stress-repressed
stress-resistent
undetectable

43
Q

classification of glycoenzymes

A

(1) Glycosyltransferases (enzymes catalyzing sugar transfer (sugar nucleotide donor-> substrate) synthesizing glycoproteins/glycoconjugates
(2) Glycosidases (endoglycosidases catalyze internal glycosidic cleavage linkage in glycans while exo cleave a monosacch from term end of glycan)
(3) Glycan-degrading enzymes(type of glycosidases destroying polysaccharides, cellulose, chitin, heparin).

44
Q

Special case

A

O-GlcNAcylation (protein glycosylation in cells in cytokines, nucleus,mito and associated w/cellular stress response).
OGT+OGA drives it.

45
Q

hexosamine biosyntehtic pathway

A

2-5% glucose fluxes Into pathway thru conversion of Fru6P-> GLCN-6P by rate limiting enzyme GFAT = UDP-GLCNA that can be recycled back to 6P by salvage pathway

all organs show O-glcNAcylated proteins. Most in brain/liver, least in bone, saliva, gall,urine. High levels =chronic aging diseases. Prolonged elevation= diabetes. blocking = no cancer progression. decreased in brain = Parkinson’s/alheimers. elevated in response to stress. can regulate protein trafficking/turnover

46
Q

Nucleus

A
  1. nucleolus (largest compartment, 25% of nucleus)
  2. stress bodies (formed from heat stress and main site of HSF1/2)
  3. bodies (regions of highest conc. of spherical rna in nucleus)
  4. nucleoli (regulates cell stress and ribosomal biogenesis that require:)
  5. pre-rRNA transcription, its processing and assembly. All localized in FC(has rRNA seq, rna polym, UBF), DFC (Ffibrillarin+Nop58), and GC (NPM1, 40s, 60s ribosomal subs).
47
Q

rRNAs and ribosomes

A

80% of RNA is rRNA. 15% tRNA, 5%mRNA+non-coding. made of 4 rRNA and up to 80 proteins in large or small subunits

small(40s): sings rRNA18s w/33 proteins

large (60S): 3rRNAs (28s, 5.8S, 5S combined w/47 proteins)

assembled(80s): 2 subunits, functions as protein synthesizing

48
Q

Eukaryotic pre-rRNA transcript

A

arranged in long tandem arrays 5’ -> 3’ order: 18S -> 5.8S -> 28S
functions was nucleolar organizer and only diff is spacer size that binds together rRNA

49
Q

chemical mod and nucleolytic processing of pre-rRNA in DFC

A

pre-rRNA is methylated and pseudouridination using small nucleolar RNA

snoNA (stem loops) associate with specific proteins to form small snoRNP with classes:
a) BoxC+D snoRNA w/ fribrillaarin mediating methylation of specific ribose
b) box H+ACA w/ dyskerin which converts uridine to pseudourine

50
Q

ribosomal subunit assembly

A
  1. specific ribosomes and snoRNAs associate w/pre-rRNA transcript and pre90S
  2. pre90S is cleaved into 60S and 40S processed in nucleoplasm
  3. small subunit processing(5min), large(30) and uses a lot of energy (GTPase+ATPase) and passes thru pore complex to cytoplasm
51
Q

cell-cycle dependent nucleolar dynamics

A

nucleoli formed around NORs on short arm of chromosomes.

m-phase: nucleolus desembles, UBF associated w/rDNA as binding platform for RNA pol 1)

G1: FC/DFC/DC evolve forming large nucleoli w/ all active NORs

s-phase: RNA Pol 1 leave rRNA preventing collision replication of machinery. Sufficient ribosomes save energy for protein synthesis rather than make new ones

G2: large fused nucleoli have inactive rDNA. Duplicated but not all active for mitosis.

52
Q

stability and stress-induced remodelling of nucleolar architecture

A

aluRNA is the glue to forming nucleolus and encoded by introns. most abundant 10% of human genome. synthesized by RNA POL II and III

under stress: cells are treated w/amanitin +induced aluRNA depletion = dispersed nucleolus

stress causers: environment can induce IGS which targets proteins w/peptide code
- accumulation of IGS = detention centre, remodelling, and nucleoli inactivation

53
Q

malfunction role of nucleolus

A

30% of proteins are involved in ribosome subunit biogenesis
70% not related but important for stress signalling
nucleolus is key hub for stress and relies on dynamic binding/release of proteins (p53-tumour suppressor gene)

54
Q

p53 signalling pathway

A

E3 ubiquitin ligase MDM2 keeps p53 levels low but stress binds MDM and activates ATM that stabilizes p53. its tetramers can now bind to p53-induced genes involved in apoptosis/division/repair

RPL26 also bind to p53 increases its levels

Molecular Stress (4 decks)

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