1. Tau
1.1. stabilize microtube
1.2. more in neuron, less in astrocyte or oligodendrioctye
1.2.1. SY5Y
1.2.1.1. detectable Tau-1, PHF-1, total Tau
1.2.1.2. undetectable:AT8, AT180, CP13 and AT270,
1.2.2. HEK
1.2.2.1. no endogeners Tau
1.2.2.2. no detectable, 1220experiment
1.2.3. Hippcampal neuron cell line
1.2.3.1. ?
1.3. 6 isoforms
1.4. 79 Ser/Thr site in longest isofrom
1.4.1. normal Tau has 30 sites phosphorylation
1.5. hyperphosphorylation
1.5.1. paired helical filament (PHF) form is one of hyperphosphorylation form
1.5.1.1. abundant in AD
1.5.1.2. tangles self-assembly
1.5.2. p-Tau: hyperphosphorylation
1.6. antibody
1.6.1. Pierce MN1020 : AT8 (M)
1.6.1.1. PHF-Tau: Ser202/Thr205
1.6.1.2. 79kD
1.6.2. abcam ab32057: anti-Tau E178clone (R)
1.6.2.1. P-Tau S396
1.6.2.2. 79kD
1.6.3. Pierce MN1000: normal Tau
1.6.3.1. normal tau
1.6.3.2. 79kD
2. fractionation
2.1. rough ER, smooth ER
2.1.1. rER: synthesize protein
2.1.1.1. PDI, ERp29, Hsp70 family(calnexin) to correct protein folding
2.1.2. rER: glycosylation
2.1.2.1. N-linked in rER
2.1.2.2. O-linked in Golgi
2.1.3. sER: synthesize lipid
2.2. gradient exp. depend on density
2.3. diff-centrifuge depend on size
2.3.1. heavy membrane:
2.3.1.1. rER (Sec61a)
2.3.1.1.1. PDI
2.3.1.1.2. calnexin
2.3.1.2. mitochondria-associated membrane (MAM:ACAT1)
2.3.2. light membrane
2.3.2.1. Golgi (beta-COP)
2.3.2.2. ER-Golgi intermediate compartment(ERGIC: ERGIC53)
3. calsenilin-info
3.1. Caspase-3 cleavage
3.1.1. "Phosphorylation of calsenilin at Ser63 regulates its cleavage by caspase-3"
3.1.1.1. Ref.
3.1.1.1.1. the heterodimer of PS N-/C-TF have functions.
3.1.1.1.2. identify of CALP/KChIP4
3.1.1.1.3. caspase-3 cleavege of AD associated protein
3.1.1.1.4. apoptosis or PS mutation increase Ca2+
3.1.1.1.5. calsenilin enhances apoptosis
3.1.1.2. Result
3.1.1.2.1. phosphorylation at Ser63 inhibits cleavage but not Ser65
3.1.1.2.2. Intracellular Ca2+ increase Ser63-p
3.1.1.2.3. phospho-site is not conserved in KChIPs
3.1.1.2.4. invivo,calsenilin is phosphorylation
3.1.1.3. Suggestion
3.1.1.3.1. apoptosis increase Ca2+
3.2. affecting PS trafficing
3.2.1. "calsenilin is degraded by the ubiquitin-proteasome pathway"
3.2.1.1. Background
3.2.1.1.1. lysosome VS. proteasome
3.2.1.1.2. calsenilin increase in AD brain
3.2.1.1.3. ubiquitin-proteasome degradation
3.2.1.1.4. proteasomal activity was decrease in AD brains
3.2.1.1.5. overexpression of calseilin enhances gamma-secrtease activity.
3.2.1.1.6. Ca2+ & PS-mutant & AD
3.2.1.1.7. PS2 overexpression induce calsenilin ER/golgi localiztion.
3.2.1.1.8. calsenilin overexpression induced kv4.2 Kchannels redistribution
3.2.1.2. Result
3.2.1.2.1. lysosome(by CQ), fusion of phagosomes and lysosomes(by NH4Cl) not affect.
3.2.1.2.2. proeastome inhibitor (by MG132) increase Calsenilin
3.2.1.2.3. calsenilin is ubiquitinated
3.2.1.2.4. MG132 increase calsenilin in ER/Golgi
3.2.1.2.5. Calsenilin tranfection + MG132 induces PS1 to ER.
3.2.1.3. discussion
4. calsenilin localiztion
4.1. KChIP4a,KIS domain
4.1.1. Introuction
4.1.1.1. Kv4 N-terminus
4.1.1.1.1. ER retention signal
4.1.1.1.2. endogenous inactivation particle
4.1.1.1.3. binding to KChIPs
4.1.1.2. KChIP4a-Bound Kv4 channel
4.1.1.2.1. traffice poorly to cell surface
4.1.1.2.2. stay in ER or Golgi apparatus
4.1.1.3. KIS domain
4.1.1.3.1. k channel inactivation suppressor
4.1.1.3.2. 34aa N terminus for KChIP4
4.1.1.3.3. conserved hydrophobic segment
4.1.1.4. localiztion
4.1.1.4.1. COS-1 cell
4.1.1.4.2. KChIP3
4.1.1.4.3. KChIP1
4.1.1.4.4. KChIP2
4.1.2. Result
4.1.2.1. KChIP4a-NTF-GFP(KISD-GFP)
4.1.2.1.1. excluded from nucleus
4.1.2.1.2. restricted to membrane network, particularly the perinuclear membranes
4.1.2.1.3. the same to KChIP2x,KChIP3x-NTF-EGFP(KISD-GFP)
4.1.2.1.4. KChIP4e -NTF,
4.1.2.2. KISD-protein
4.1.2.2.1. KChIP4a, KChIP2x, KChIP3x, DPP6-S, DPP6a
4.1.2.3. KIS element
4.1.2.3.1. encodes Type I transmembrane domain
4.1.3. Disucssion
4.1.3.1. multiple KChIP isoforms have KISDs
4.1.3.2. the KISD contains a transmembrane segment
4.1.3.3. the KISD transmembrane segment mediate ER retenition
4.1.3.4. NMR analysis with cell-free, lipid-free conditions
4.1.3.4.1. KISD hydrophobic domain could bind to the KChIP core in an auto inhibitory manner
4.1.3.4.2. but in cellular , they suggested KISD inserts itself into the lipid bilayer than interacting with itself
4.2. Subcellular localization
4.2.1. introduction
4.2.1.1. KCHIP alternative splicing
4.2.1.1.1. Introduction
4.2.1.1.2. Result
4.2.1.1.3. discussion
4.2.1.2. controversial localization of KChIP3
4.2.1.2.1. peirnuclear
4.2.1.2.2. nuclear
4.2.1.2.3. intracellular membrane and the cell membrane
4.2.1.3. KChIP2,3 to membrane by palmitoylation
4.2.1.3.1. Result
4.2.1.3.2. discussion
4.2.1.4. KChiP1 to post-ER transport vesicles by myristoylation
4.2.1.5. COS-1/7 cell without kv4
4.2.1.5.1. diffuse distribution all over the cell including nucleus for Kchips
4.2.1.6. kchip3 function as DREAM is unique
4.2.1.6.1. primarily regulated by calcium binding
4.2.1.6.2. C-terminal part binding to DNA as DRE
4.2.1.6.3. DRE: downstream regulatory element
4.2.2. Result
4.2.2.1. KChIP1a(lb[II]), 1b(lb), la[II]
4.2.2.2. KChIP1
4.2.2.2.1. Ia(p)
4.2.2.2.2. KChIP1b - Ib(m)-II
4.2.2.2.3. KChIP-la[II]
4.2.2.3. KChIP2
4.2.2.3.1. KChIP2L (la)
4.2.2.3.2. KChIP2 (la[IIb])
4.2.2.3.3. KChIP2S (la[llab])
4.2.2.3.4. KChIPg (lb[llab])
4.2.2.4. KChIP3
4.2.2.4.1. Depended on the N-terminus
4.2.2.4.2. KChIP3 (la) (with S-palmitoylation site)
4.2.2.4.3. KChIP3-lb (transmembrane helix, signal peptide)
4.2.2.5. KChIP4
4.2.2.5.1. different N-termini had divergent effects
4.2.2.5.2. KChIP4bl (la)
4.2.2.5.3. CALP216 (la[ll])
4.2.2.5.4. KChIP4-lb[ll]
4.2.2.5.5. KChIP4a (ld[ll])
4.2.2.5.6. KChIP4-le[ll]
4.2.2.6. endogenous
4.2.2.6.1. no KChIP in nuclei of rat primary cortical neurons
4.2.3. discussion
4.2.3.1. KChIP1, KChIP4-lb[ll]
4.2.3.1.1. similar punctate cytoplamic
4.2.3.1.2. post-ER transport vesicles
4.2.3.1.3. KChIP1 by N-myristoylation
4.2.3.2. CRE-dependent transcription
4.2.3.2.1. none of KChIP interfere with CRE-dependent transcription in neurons
4.2.3.2.2. no interaction of KChIP3-Ia with CREB by EMSA
4.2.3.2.3. BDNF is not regualted by KChIPs in primary coritcal neurons
4.2.3.2.4. CREB is a central TF in calcium-regulated processes in neurons
4.2.3.2.5. previous studies on KChIP3 were usage of 30 additional N-terminal acids not present in the WT.
5. APP
5.1. minreview
5.1.1. APP695, 751, 770 isoforms
5.1.1.1. APP695 in neurons
5.1.1.2. APP751 in non-neurons
5.1.2. three WB band
5.1.2.1. N-/O-glycosylatied
5.1.2.2. sulfated
5.1.3. Transport
5.1.3.1. ER:N-glycosylation (imAPP)
5.1.3.1.1. Golgi:O-glycosylation(mAPP)
5.1.3.1.2. two different O-glycosylation
5.1.4. proteolytic processing
5.1.4.1. secretory pathway
5.1.4.2. on the PM
5.1.4.3. in the endocytotic cycle
5.1.4.4. most APP is degradated by lysosomes
5.1.4.4.1. introduction
5.1.4.4.2. Result
5.1.4.4.3. suggesting
5.1.5. occur in various sites
5.1.5.1. secretory pathway
5.1.5.2. endocytic pathwyas
5.1.6. gamam-secretase independent degradation
5.1.6.1. lysosometropic drug chloroguqine
5.1.6.1.1. PC12 + APP695, 751, 770
5.1.6.1.2. lysosomal APP processing
5.1.6.1.3. Chloroquine elevated PH of lysosome.
5.1.6.1.4. chloroquine has no effect on APP maturation, APP secretion
5.1.6.1.5. chloroquine increase matured APP and APP-CTF
5.1.6.1.6. APP-CTF reached max at 1h. and slowly decrease
5.1.6.2. acidic degradation
5.1.6.2.1. Leupeptin, E-64, chloroquine
5.1.6.2.2. HEK293 + APP751/695
5.1.7. amyloidogenesis
5.1.7.1. a-secretase
5.1.7.1.1. ADAM: biosynthesized ADAM transported to golgi
5.1.7.1.2. ADAM10
5.1.7.1.3. ADAM17
5.1.7.2. b-secretase
5.1.7.2.1. BACE
5.1.7.3. gamma-secretase
5.1.7.3.1. PS
5.1.7.3.2. nicastrin(NCT), APH-1, PEN-2
5.1.7.3.3. well ordered fashion during membrane trafficking through the ER, Golgi to form complex
5.1.7.4. processing position
5.1.7.4.1. after transition through the golgi
5.1.7.4.2. and in endosome compartment
5.1.8. APP cytoplasmic tail interaction (695aa)
5.1.8.1. 653-656aa: YTSI
5.1.8.2. 667-672aa: VTPEER
5.1.8.2.1. a-helical structure
5.1.8.3. 681-687aa: GYENPTY
5.1.8.3.1. recognized by proteins containing PID (phosphotyrosine interaction domains)
5.1.8.4. YENPTY: promote clathrin-mediated endocytosis
5.1.8.4.1. NPXY is interalization signal
5.1.8.5. binding patterner
5.1.8.5.1. FE65-AICD: gene transactivation by AICD
5.1.8.5.2. X11 and X11L-APPCTF
5.1.8.5.3. JIP1b-APP
5.1.8.5.4. SorLA, LR11
5.1.9. ubiquilin
5.1.9.1. ubiquilin-1 regultes trafficking and processing
5.1.9.1.1. intro
5.1.9.1.2. result
5.1.9.2. ubiquilin-1 & APP
5.1.9.2.1. ubiquilin-1 sequester APP in golgi
5.1.9.2.2. delay its assess to PM and proteolytic processing
5.1.9.2.3. ubiquilin-1 stimulating APP lysine63-linked polyubiquitination
5.1.9.2.4. K668R abrogated ubiquitination and golgi sequestertion
5.1.10. JNK & APP phosphorylation
5.1.10.1. AICD (APP intracellular domain)
5.1.10.1.1. important for metabolism
5.1.10.2. Y653, S655, T668, S675, Y682, T686, Y687 are phosphorylated in AD