Autophagy and Apoptosis

8/18/2019 Autophagy and Apoptosis

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PMID: 24679527

• The process of macroautophagy (herein, autophagy is !est un"erstoo" in the conte#t of

nutrient star$ation (%roemer et a&', 2)* Mi+ushima an" %omatsu, 2))' hen energy

in the form of -TP is &imiting, -MP .inase (-MP% !ecomes acti$e, an" this can "ri$e

autophagy' Depri$ation from gro/th factors an"0or amino aci"s &ea"s to the inhi!ition of T13), /hich, /hen acti$e, represses con$entiona& autophagy'

• "ou!&emem!rane structure, the autophagosome, fuses /ith &ysosomes to "e&i$er the

contents for "egra"ation, an" this in$o&$es a proteo&ipi" mo&ecu&e, 3II, a componentof the autophagosome compose" of a protein, 3, an" a &ipi",

phosphati"y&ethano&amine' 3II is generate" !y a process resem!&ing u!iuitination,

in$o&$ing 8), 82, an" 8 &igases'

• 3I, is generate" !y the action of a protease, -T4, /hich c&ea$es 3 to pro"uce

3I' This is !oun" !y the 8), -T7, an" transferre" to the 82, -T' The 8 &igase is

a comp&e# compose" of -T)6 an" -T)25* the &atter is pro"uce" !y another reactionin /hich -T)2 is !oun" !y the 8), -T7, transferre" to a "ifferent 82, -T), an"

from there to -T5' The process !y /hich -T)25 is forme"an", su!seuent&y, 3

II (a&so .no/n as 3P8 is generate"is referre" to as the e&ongation reaction an" is

reuire" for the formation of the autophagosome'

• Initiation process reuires the action of the 3&ass III PI .inase, ;P


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• Due to opposite effects of c&ass I an" c&ass III PI%s on autophagy, M- can re"uce or

promote autophagy "epen"ing on the re&ati$e acti$ation state of the t/o path/ays'

• M- a&so has "irect effects on g&ucose0g&ycogen meta!o&ism in"epen"ent of its

autophagymo"u&ating effects an" e&e$ates &ysosoma& p@ in &i$ing hepatocytes, !ut not

iso&ate" &ysosomes'

• noncanonica& !ec&in )in"epen"ent autophagy0mitophagy contri!utes to MPPA to#icity,

as sho/n !y B- interference .noc."o/n of -tg7 an" 30-tg>'

• =&unting the autophagy response has a&so !een sho/n using "ominant negati$e ;ps4 to

pre$ent hy"rogen pero#i"eme"iate" &ysosoma& &ea.age an" caspase acti$ation'

• Par.in trans&ocation to C33P"epo&ari+e" mitochon"ria resu&ts in their e$entua& c&earance

through -tg5"epen"ent mechanisms'

• Trans&ocation of tagge" par.in to mitochon"ria an" its u!iuitinating acti$ity is essentia&

for enhance" mitochon"ria& autophagy in C33P0333Ptreate" ce&&s' @o/e$er, par.in

monou!iuitination of =c&2 enhances the a!i&ity of =c&2 to !in" !ec&in ) an" suppress

autophagy'

• -tg 2 !in"s to -tg)), a .no/n a"aptor protein for se&ecti$e autophagy in yeast' This

system recruits mitochon"ria to autophagosomes, !ut "oes not "irect&y regu&ate

macroautophagy in"uction itse&f'

• -tg 2 !in"s to -tg)), a .no/n a"aptor protein for se&ecti$e autophagy in yeast' Thissystem recruits mitochon"ria to autophagosomes, !ut "oes not "irect&y regu&ate

macroautophagy in"uction itse&f'

• - .ey feature of ua&ity contro&re&ate" autophagy re&ates to the a!i&ity of the ce&&u&ar

autophagy machinery to se&ecti$e&y remo$e "amage" proteins an" organe&&es /hi&e

sparing their norma& counterparts'

• par.in functions on&y in the cargo recruitment step of mitophagy, !ut other mechanisms

in$o&$ing Bi# me"iate the in"uction of autophagy !y "epo&ari+ation' Par.in has a&so !een

reporte" to po&yu!iuitinate $o&tage"epen"ent anionse&ecti$e channe& protein )

(;D-3)'

• Mitophagy can !e initiate" prior to "epo&ari+ation of the mitochon"ria& mem!rane

potentia& in se$era& mo"e& systems' Mitochon"ria& "epo&ari+ation occurs "o/nstream of

autophagosome formation in reticu&ocytes "uring Bi#"epen"ent "e$e&opmenta&

mitophagy'


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• Depo&ari+ationin"epen"ent, Bi#"epen"ent mitophagy may in$o&$e "irect interactions of

Bi# /ith an 3 homo&og -=--P'

• hether or not sta!&e par.in recruitment to mitochon"ria is necessary for its

compensatory effects is un.no/n, !ut hy"rogen pero#i"e can recruit par.in to

mitochon"ria through a mechanism not reuiring mitochon"ria& targeting of PIB%)'

PMID: 2)>56>59

• Catty aci"s are chemica&&y c&assifie" as saturate" an" unsaturate" (monounsaturate" an"

po&yunsaturate", an" their structure affects their !io&ogica& functions' Pa&mitic aci" (P-,

a saturate" fatty aci", an" o&eic aci" (1-, a monounsaturate" fatty aci", are t/o of the

most a!un"ant fatty aci"s present in the "iet an" in serum'

•


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• Monounsaturate" 1- /as rea"i&y con$erte" to Tenriche" &ipi" "rop&ets, in"uce"

autophagy, an" /as resistant to apoptosis in @ep2 ce&&s' In contrast, saturate" P- /as

on&y s&ight&y con$erte" to Tenriche" &ipi" "rop&ets resu&ting in the in"uction of apoptosis /ithout the acti$ation of autophagy'

• There is crossta&. !et/een fatty aci"in"uce" apoptosis an" autophagy, in /hichsaturate" P-in"uce" apoptosis suppresses autophagy !y caspaseme"iate" c&ea$age of =ec&in )' 3on$erse&y, autophagy a&so attenuate" fatty aci"in"uce" apoptosis an"

accumu&ation of &ipi"s'

• num!er of &ipi" "rop&ets an" the &e$e&s of T are significant&y higher in 1-treate"

@ep2 ce&&s than in P-treate" ce&&s' -s a resu&t, 1- fai&s to in"uce apoptosis an" e$en

protects against P-in"uce" apoptosis in @ep2 ce&&s'

• rapamycin suppresses mT1 an" in"uces autophagy in $arious ce&& &ines' Inhi!ition of

mT1 &ea"s to re"uce" phosphory&ation of t/o of its "o/nstream effectors, p7


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• 8CPP-%2 /as recruite" to mitochon"ria an" co&oca&i+e" /ith =BIP after 333P

treatment'

• rotenone an" 6hy"ro#y"opamine hy"roch&ori"e (61@D-, the other comp&e# I

inhi!itors, a&so in"uce par.insonian syn"rome'

• n"er car!ony&cyani"e ch&oropheny&hy"ra+one (333P treatment, the recruitment of

P-%2 onto mitochon"ria in"uces mitochon"ria& u!iuitination an" promotes

mitophagy'

• =BIP me"iates mitophagy "epen"ent on the presence of P-%2 that u!iuitinates

=BIP on mitochon"ria, resu&ting in a recognition of u!iuitinate" mitochon"ria !y

autophagic a"aptors'

• Increase" "egra"ation of T1MM2 an" 31F4I)/as o!ser$e" in @8%29- ce&&s

transfecte" /ith C&ag=BIP or C&agP-%2, a&though there /as no "ifference in the

in"uction of autophagy'

• Co&&o/ing the 333P treatment, /e o!ser$e" a num!er of C&ag=BIP punctas that co

&oca&i+e" /ith 8CP3 in @8%29- ce&&s, in"icating that C&ag=BIP promotes the

transport of its associate" mitochon"ria to the autophagosomes'

• Minima& amounts of P-%2 coimmunoprecipitate" /ith =BIP, ho/e$er, the

interactions /ere significant&y increase" after P-%2 /as recruite" to the mitochon"ria

after the 333P treatment'


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• The 333Pin"uce" trans&ocation of B=) to mitochon"rion /as inhi!ite" in the

=BIP .noc."o/n ce&&s'

• The mitochon"ria& comp&e# I inhi!itionin"uce" =BIP "egra"ation may not through

the mitophagy path/ay'

oo. at the fo&&o/ing references : )6, )9, 25, 27, G 7

PMID: 254477

• if the mitochon"ria& mem!rane potentia& is intact, the serinethreonine .inase PIB%) is

trans&ocate" to the inner mitochon"ria& mem!rane to un"ergo proteo&ytic c&ea$age !y

P- (preseni&inassociate" rhom!oi"&i.e protein ()2, ) fo&&o/e" !y proteasoma&"egra"ation'

•

If the mitochon"ria& mem!rane potentia& is "issipate", PIB%) accrues on the outer mitochon"ria& mem!rane as its 6.Da fu&&&ength isoform to recruit cytoso&ic Par.in (2,4, 5, )5, an 8 u!iuitin &igase, /hich u!iuity&ates numerous 1MM proteins &ea"ing to

autophagosome engu&fment of the u!iuitintagge" "epo&ari+e" mitochon"ria an"

su!seuent &ysosoma& "egra"ation, i'e' mitophagy'

• -&though the &e$e& of 6.Da fu&&&ength PIB%) /as pre$ious&y sho/n to increase /hen

ce&&s are incu!ate" /ith a /or.ing concentration of 333P (4, )2, /e sho/ that this

increase can on&y !e e#presse" /hen a thresho&" concentration of g&ucose is met'

• 8#cee"ing a conser$e" g&ucose concentration thresho&" e&icits the mitophagic response

!et/een "ifferent ce&& &ines upon mitochon"ria& "amage, an" the sensiti$ity of thePIB%)0Par.in mitophagy path/ay to g&ucose is characteristic of the path/ay itse&f an"not a phenomenon e#c&usi$e to a particu&ar ce&& &ine'

• The a!i&ity of PIB%) to sense g&ucose concentration an" a&ter e#pression &e$e&s is

in"epen"ent of its .inase acti$ity'

• mB- e#pression &e$e&s for PIB%) remain &arge&y unchange" in the "iffering g&ucose

concentrations /hether or not 333P /as present'

• fu&&&ength PIB%), upon 1MM accumu&ation, is not targete" for e#cessi$e "egra"ation

/hen g&ucose is "ep&ete", an" to the contrary, the &e$e&s are sustaine"'

• Decrease in PIB%) &e$e&s seen in g&ucose /ith"ra/a& is most &i.e&y "ue to trans&ationa&

suppression'

• &ucose a&one is not a necessary $aria!&e, !ut an acti$e g&yco&ytic path/ay is necessary

for acti$ation of the PIB%)0Par.in mitophagy path/ay'


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• Mitochon"ria& &oca&i+ation of Par.in is not specific to g&ucose /hen -TP pro"uction

remains acti$e' >, 95'


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MCB)02 an" MI1 are share" PIB%) an" Par.in su!strates' They are phosphory&ate" !y

PIB%) !efore their u!iuitination !y Par.in (>>, 95'

• Par.in me"iate" "egra"ation of the fusion proteins MCB)02 maintains mitochon"ria in a

fragmente" state, /hich faci&itates mitophagy (>>'

• MI1 is a component of the comp&e# that anchors .inesin to the mitochon"ria an"

Par.inme"iate" "egra"ation of MI1 &ea"s to re&ease of the "amage" mitochon"ria

from the tu!u&in net/or.'

•


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"isco$ere" that P-M5 is reuire" for sta!i&i+ation of PIB%) on "amage" mitochon"ria

an" that &oss of P-M5 a!rogates PIB%)me"iate" mitophagy (57'

• 3ar"io&ipin is present in inner mitochon"ria& mem!ranes, /here it is essentia& for optima&

function of numerous en+ymes in$o&$e" in mitochon"ria& meta!o&ism ('

• '

Thus this suggests that the regu&ation of autophagy !y the =@on&y proteins might"epen" on their su!ce&&u&ar &oca&i+ation'

• /hen mitochon"ria& function is compromise", they pro"uce &ess -TP, /hich &ea"s to

acti$ation of -MP% an" su!seuent initiation of autophagy (Cig' = ()' -MP% phosphory&ates an" acti$ates the nc5)i.e %inases (%, /hich, in turn, acti$ates

the =83IB )0;P


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• =BIP in"uces autophagy an" mitophagy in"epen"ent&y of the mPTP (74' This suggests

that the mPTP is responsi!&e for acti$ating mitophagy un"er certain con"itions such as

star$ation' It is current&y un.no/n if the mPTP p&ays a ro&e in PIB%)0Par.inme"iate"mitophagy'

• -M=-) (acti$ating mo&ecu&e in =ec&in)regu&ate" autophagy is a critica& regu&ator of autophagy initiation an" interacts /ith =83IB ) to initiate formation of the phagophore(2' -M=-) a&so interacts /ith Par.in at the mitochon"ria, an" the interaction

!et/een Par.in an" -M=-) is increase" "uring pro&onge" mitochon"ria&

"epo&ari+ation,-M=-) is not reuire" for trans&ocation of Par.in to "epo&ari+e"mitochon"ria, !ut it is critica& for su!seuent mitochon"ria& c&earance (>4'

• -M=-) can a&so interact "irect&y /ith 3 through its I (3 interacting region

motif, an" this interaction is critica& for Par.in"epen"ent mitophagy (>4' ' This a&ternati$e autophagy path/ay p&ays aro&e in c&earing mitochon"ria "uring erythrocyte "ifferentiation (29'

• Microautophagy is another &ess .no/n form of autophagy that occurs in mamma&ian

ce&&s' In this process, proteins an" organe&&es are "irect&y interna&i+e" into &ysosomes

through in$aginations of the &ysosoma& mem!rane' Microautophagy has primari&y !een

stu"ie" in yeast, !ut there is e$i"ence that it a&so ta.es p&ace in mamma&ian ce&&s (65, >7'

• PIB%)"eficiency increase" the suscepti!i&ity of the heart to I0 in?ury e# $i$o (5'

Increase" mitophagy /as initia&&y "escri!e" in myocytes o$ere#pressing =BIP an" in

hearts su!?ecte" to e# $i$o I0 (26'

• Par.in"eficient mice accumu&ate "ysfunctiona& mitochon"ria after a myocar"ia&

infarction, /hich &ea"s to increase" morta&ity (5' Par.in "eficient mice are a&so more

suscepti!&e to "o#oru!icinme"iate" car"ioto#icity(2' -&though enhance" autophagy is protecti$e "uring ischemia, it s/itches to a "etrimenta& ro&e "uring reperfusion (62'

• Par.inme"iate" mitophagy is protecti$e in pancreatic ce&& function in "ia!etes ('

Dia!etic car"iomyopathy is associate" /ith mitochon"ria& "ysfunction (6, an" it is possi!&e that impaire" autophagy an" mitophagy may contri!ute to the patho&ogy'

• Interesting&y, Fu et a&' (97 reporte" that genera& autophagy, as /e&& as PIB%) an" Par.in

protein &e$e&s, are significant&y re"uce" in Type ) "ia!etic hearts' Interesting&y, this stu"y

note" that a!9 is increase" an" that it co&oca&i+es /ith mitochon"ria, suggesting that


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• @o/e$er, the &oss of par.in "i" not "ecrease mitochon"ria& u!iuitination an"

e#acer!ate" "efects in !oth mitochon"ria& an" car"iac functions'


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• Mitochon"ria& structure an" function /ere a&tere", apoptosis /as not in"uce" in Drp)%1

car"iomyocytes' Immuno!&otting sho/e" no proteo&ytic acti$ation of caspase or

c&ea$age of its su!strate P-P in Myh6Drp)%1 hearts'

• p62 is "egra"e" !y autophagy an" inhi!ition of autophagy increases its a!un"ance

(%&ions.y et a&, 2)2 (Tana.a et a&, 2)' The &e$e& of p62 /as increase" Kfourfo&" inMyh6Drp)%1 compare" /ith contro& an" @et &ittermates (Cig 4-' In contrast, 3II&e$e&s remaine" unchange"'

• p62 signa&s /ere concentrate" in punctate structures "ecorate" !y u!iuitin, /hich is

con?ugate" to mitochon"ria& proteins "uring mitophagy (ou&e J Baren"ra, 2))'

• To test /hether these structures contain mitochon"ria, /e immunostaine" heart sections

/ith anti!o"ies to p62, u!iuitin, an" pyru$ate "ehy"rogenase (PD@, a mitochon"ria&matri# protein' Many &ysosomes /ere foun" near u!iuitin"ecorate" mitochon"ria' It

appears that mitophagy /as incomp&ete&y e#ecute" in Drp)%1 car"iomyocytes,

generating interme"iate structures /ith accumu&ate" p62 an" u!iuitinate" proteins onmitochon"ria /ithout increases in the amount of 3II'

• To test /hether the accumu&ation of p62, in the a!sence of Drp), is "ue to inhi!ition of

autophagic f&u#, /e e#amine" the accumu&ation of 3II in T an" Drp)%1 mouseem!ryonic fi!ro!&asts (M8C in the presence of !afi&omycin -, e foun" that 3II

simi&ar&y !ui&t up in !oth ce&&s, suggesting that the &oss of Drp) "oes not affect autophagic

f&u#'

• hi&e Drp)%1 an" Par.inDrp)%1 hearts ha" "ecrease" Drp) &e$e&s (Cig 5-, Par.in%1

hearts sho/e" increase" Drp) &e$e&s, consistent /ith a pre$ious stu"y sho/ing that

par.in u!iuitinates Drp) for "egra"ation (ang et a&, 2))'

• The a!un"ance of p62 /as simi&ar&y increase" in Drp)%1 an" Par.inDrp)%1 hearts' In

contrast, Par.in%1 hearts containe" norma& &e$e&s of p62' Curthermore,

immunof&uorescence of heart sections sho/e" that p62 /as accumu&ate" in !oth Drp)%1an" Par.inDrp)%1 hearts, !ut not in contro& an" Par.in%1 hearts'

• 3o&oca&i+ation of p62 an" u!iuitin /as a&so unaffecte" in Par.inDrp)%1 hearts'

Increase" &e$e&s of p62 an" mitochon"ria& u!iuitination are in"epen"ent of par.in inDrp)%1 hearts'

• ; function significant&y /orsene" in Par.inDrp)%1 mice compare" /ith Drp)%1

mice, /ith a "rop of heart contraction from K45L (Drp)%1 to KL (Par.inDrp)%1'


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Tim2 /as unaffecte"' Thus, par.in is critica& for car"iac function an" the maintenance

of the e&ectron transport chain in car"iomyocytes /hen Drp) is a!sent'

• That par.in "i" not accumu&ate in p62 an" u!iuitin"ecorate" structures or en&arge"

mitochon"ria in Myh6Drp)%1 hearts'

• e foun" that Par.in%1::7 Drp)%1 mice sho/e" acce&erate" PB (Pur.en?ie Beurons

"egeneration compare" /ith 7Drp)%1 mice' The mitochon"ria in 7Drp)%1 mice

/ere e&ongate" "ue to unoppose" fusion in PBs an" then further change" their

morpho&ogy to &arge spheres, &i.e&y "ue to o#i"ati$e "amage'

• The en&arge" mitochon"ria in 7Drp)%1 PBs accumu&ate" u!iuitinate" proteins'


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PMID: 256644

• The re"uction of ∆Ψm &ea"s to the accumu&ation an" acti$ation of PIB%) in the

mitochon"ria ()2, )7, /hich &ea"s to the phosphory&ation of a &atent form of Par.in, priming its 8 acti$ation ()7, )>' PIB%) a&so phosphory&ates u!iuitin ()9 G2), /hich

in turn fu&&y acti$ates Par.in 8 acti$ity, &ea"ing to Par.in trans&ocation from the cytoso&to the mitochon"ria an" the su!seuent u!iuitination of mitochon"ria& proteins ()4, )5'

• The u!iuitination of mitochon"ria& proteins main&y pro"uces ys6&in.e"

po&yu!iuitin an" on&y a sma&& portion of ys4> &in.ages (22, 2' The ys6&in.e"

po&yu!iuitin chain is propose" to acti$ate PIB%) (24 an" the mitochon"ria&

trans&ocation of Par.in (25'

• !c) is an 82 en+yme crucia& for generating ys6&in.e" chains ('

• In the !c) mutant ce&&s har!oring the &o#Pf&an.e" !c) gene, !c) can !e

inacti$ate" !y Do#in"uce" f&o#out' e inacti$ate" !c) !y Do# treatment an"

in"uce" the mitochon"ria& trans&ocation of CPPar.in an" the accumu&ation of u!iuitin

chains using 333P' The mitochon"ria& trans&ocation of CPPar.in occurre" /ith simi&ar

efficiency (Cig' ), - an" =' In contrast, the accumu&ation of tota& u!iuitin (Cig' ), 3 an"8 as /e&& as ys6&in.e" po&yu!iuitin (Cig' ), D an" 8 in the mitochon"ria /as

"ramatica&&y re"uce" in the a!sence of !c) acti$ity'

• hen Par.in is acti$ate" upon 333P treatment, Par.in is su!?ecte" to auto"egra"ation

!y the proteasome ()>' The "egra"ation efficiency of @-tagge" Par.in /as simi&ar

!et/een !c)A0A an" !c)0 M8Cs, suggesting that the formation of ys6&in.e" po&yu!iuitin affects neither the acti$ation of Par.in nor the autophagic c&earance of mitochon"ria'

• It has !een propose" that ys6&in.e" u!iuitination of PIB%) !y T-C6 is reuire"

for the mitochon"ria& accumu&ation of PIB%) an" mitochon"ria& trans&ocation of Par.in

upon a re"uction of Qm (24' PIB%) sta!i&i+ation on the mitochon"ria& outer mem!rane

stimu&ates its "imeri+ation an" is c&ose&y corre&ate" /ith its autophosphory&ation at an"


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u!iuitination &i.e&y me"iates the recruitment of autophagyre&ate" proteins, as propose"

in stu"ies of mitophagy ()4, 22'

• mitophagy an" #enophagy suggest that the autophagy machinery can recogni+e other

po&yu!iuitin &in.ages in a""ition to ys6 or that ys6 &in.age is not in$o&$e" in this

step' -&though ys6&in.e" u!iuitination is not essentia&&y reuire" for mitochon"ria&trans&ocation of Par.in, the inhi!ition of a&& of u!iuitination reactions !y an 8)specific

inhi!itor comp&ete&y suppresses Par.in trans&ocation, suggesting that u!iuitination is

part of the regu&ation in Par.in trans&ocation'

PMID: 227477

• The 3rohnHs "isease ris. a&&e&es resu&t in "ecrease" autophagic function, suggesting that

autophagy is reuire" to &imit the intestina& immune response'

• -utophagy is in$o&$e" in the .i&&ing of intrace&&u&ar pathogens !y phagocytes, inc&u"ing


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D an" periphera& I3-M) is consistent /ith increase" formation of an immuno&ogic

synapse'

• In an a&&ogeneic pro&iferation assay, -T)6)&o/ D3s in"uce" significant&y more

pro&iferation than contro& D3s, suggesting increase" immunogenicity' %noc."o/n of

-T)6) in murine =MD3s resu&te" in increase" pro&iferation of o$a&!uminspecific Tce&&s in the presence of their cognate antigen'

• 8#pression of acti$ation mar.ers inc&u"ing @-D, 3D>, 3D>6, an" 3D> /as not

a&tere" after .noc."o/n' Curthermore, "ecrease" e#pression of -T)6) "i" not affectthe &e$e&s of proinf&ammatory cyto.ines inter&eu.in (I), I6, an" tumor necrosis

factor, or of the regu&atory cyto.ine I), either in the presence or a!sence of

a&&ogeneic T ce&&s'

• =oth -T)6)&o/ an" IM&o/ D3s in"uce" significant&y more I)7Gpro"ucing

ce&&s' In contrast, the &e$e& of interferonUGpro"ucing ce&&s /as not a&tere"'

• 3ognate D3GTce&& interactions %=) is recruite" to the immuno&ogic synapse an"

acti$ates -MP%' The ensuing inhi!ition of mT1 acti$ity then initiates autophagic

acti$ity'

• 3ognate interactions !et/een D3s an" T ce&&s &ea" to %=)G-MP%Gme"iate" inhi!ition

of mT1 signa&ing, /hich resu&ts in autophagosome formation' The autophagosomes

contain synaptic components an" are reuire" to &imit !oth the "uration of synapseformation an" the magnitu"e of the ensuing Tce&& acti$ation'

• -utophagy contri!utes to innate immunity through "egra"ation of intrace&&u&ar

pathogens, inc&u"ing Myco!acteria an" < enterica' Diminishe" autophagy therefore mayresu&t in "efecti$e innate immunity, e#cessi$e micro!ia& e#pansion, an" un!a&ance"

a"apti$e immune acti$ation'

• %=) is recruite" to the periphery of the D3GTce&& interface, supporting an acti$e ro&e

for the protein in ce&&u&ar interactions'


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• In ce&&s star$e" for > h an" e#pressing ye&&o/ f&uorescent protein (CPtagge" -MP)

(-MP)GCP or staine" for en"ogenous -MP), /e o!ser$e" tu!u&ar structures

e#ten"ing from auto&ysosomes'

• 1!ser$e" -MP) on the surface of the tu!u&es !ut sho/e" that they containe" &itt&e or

no "iscerni!&e &umena& content from the auto&ysosomes'

• tu!u&e e#tension is a continuous an" high&y "ynamic process /ith the "ista& portions of

tu!u&es e#tru"ing free $esic&es photoacti$ata!&e CPtagge" -MP) (P-CPG-MP)

for pu&sechase ana&ysis of the auto&ysosoma& mem!rane' -fter 4 h of star$ation,in"i$i"ua& auto&ysosomes /ere &aseracti$ate" an" fo&&o/e" !y time&apse microscopy'

ithin 2 min, /e o!ser$e" tu!u&e formation fo&&o/e" !y !u""ing of -MP)) $esic&es'

• -MP)) tu!u&es /ere not staine" !y the aci""epen"ent "ye ysotrac.er, "espite strong

staining of the auto&ysosomes' The &ysosoma& su!strate DE=


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• 1$ere#presse" constituti$e&y acti$e a!7, /hich is permanent&y associate" /ith the

mem!rane, an" foun" that - /as a!rogate", resu&ting in en&arge" an" &ong &astingauto&ysosomes' Inhi!ition of - !y rapamycin !&oc.s "issociation of a!7 from the

"isten"e" auto&ysosomes it pro"uce", in"icating that mT1 might regu&ate - through

a!7'

• inhi!ition of auto&ysosoma& protein "egra"ation !y the &ysosoma& inhi!itors &eupeptin or

8640pepstatin - "uring star$ation a!o&ishe" reacti$ation of mT1' Inhi!ition of

&ysosoma& proteases a&so a!rogate" the attenuation of autophagy an" -, resu&ting inen&arge" an" &ong&asting auto&ysosomes'

• fi!ro!&asts "eri$e" from patients /ith the &ysosome storage "iseases (


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su!stantia& co&oca&i+ation of !oth proteins in star$ation/ith or /ithout ch&orouine

an" in the presence of rapamycin'

• D1 "i" not co&oca&i+e /ith &ysosoma&associate" mem!rane protein ) (-MP)CP or

/ith ysotrac.er un"er any of the stu"ie" con"itions, /hich in"icates that D1 is a

mar.er of ear&y autophagosomes'

• D1 physica&&y interacts /ith components of the autophagy machinery such as 3 or

o&giassociate" -TPase enhancer of )6 .Da (-T8)6'

• -mino aci" star$ation cause" a mar.e" increase in protein "egra"ation (52L increase,

an" D1 gainoffunction together /ith star$ation further acti$ate" this process' D1

increase" the num!er of autophagosomes !oth un"er !asa& con"itions an" amino aci"star$ation'

• D1 gainoffunction "i" not a&ter the ce&& $ia!i&ity ana&yse" /hen assaye" un"er !asa&

con"itions or after autophagy acti$ation ("ata not sho/n' - B8< motif /as i"entifie" inD1 (resi"ues 2G4, an" the mutation (6-0I7-0I>-04- nu&&ifie" the capacityof D1 to &ea$e the nuc&eus in response to stress'

• D1 .noc."o/n cause" a mar.e" "ecrease in 3positi$e puncta un"er !asa&

con"itions (45L re"uction an" "uring star$ation' D1 repression a&so cause" a "ecrease

in the num!er of autophagosomes (75L re"uction an" a su!stantia& inhi!ition of protein

"egra"ation'

• "D1 is pre"icte" to interact /ith -tg>a an" -tg>! !y highthroughput t/ohy!ri"

screening, suggesting that the interaction !et/een D1 an" -tg> is conser$e" from

humans to f&ies'

PMID: 22257>>

• It has !een reporte" that u!iuitinate"protein aggregates /ere forme" in pancreatic

ce&&s "uring o#i"ati$e stress associate" /ith hyperg&ycemia an" /ere regu&ate" !y

autophagy'

• -tg7Xce&& mice sho/e" significant hyperg&ycemia, g&ucose into&erance an"

hypoinsu&inemia' Morpho&ogic ana&ysis sho/e" "ecrease" ce&& mass, a&ong /ith

increase" ce&& "eath an" re"uce" ce&& pro&iferation' Due to the re"uce" ce&& mass, pancreatic insu&in content /as a&so re"uce"'

• hen g&ucosein"uce" changes of cytoso&ic 3a2A concentrations (R3a2ASc /ere

measure" in $ia!&e is&et ce&&s, they /ere significant&y impaire" in is&ets of -tg7Xce&&mice compare" to contro& is&ets'


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• hi&e so&u!&e short&i$e" u!iuitinate" proteins are remo$e" through proteasoma&

path/ay, inso&u!&e or &arge &ong&i$e" hea$i&y u!iuitinate" proteins are preferentia&&y

c&eare" through autophagy path/ay'

• arge inc&usion !o"ies /ere forme" in autophagy"eficient ce&&s that comprise

u!iuitin, p62 an" "egenerate" proteins'

• The "ecrease" function an" mass of ce&&s of -tg7Xce&& mice might !e "ue to

mitochon"ria& "ysfunction an"0or 8 stress, !ecause mitochon"ria an" 8 are main

ce&&u&ar organe&&es re?u$enate" !y autophagy an" critica& for ce&& function an" sur$i$a&'

• Protein "isu&fi"e isomerase (PDI, an 8resi"ent protein, e#ists in !oth si"es of

mem!ranes that san"/iche" iso&ation mem!rane, suggesting that iso&ation mem!rane is

forming autophagosome cra"&e stac.e" !et/een t/o 8 mem!ranes'

• sua&&y misfo&"e" or e#cessi$e unfo&"e" proteins are c&eare" through u!iuitination an"

proteasoma& "egra"ation in the cytop&asm in a process ca&&e" 8associate" protein"egra"ation (8-D'

• Misfo&"e" protein shou&" !e unfo&"e" again an" !e transporte" across 8 mem!rane

!ac. to cytop&asm in a process ca&&e" retrogra"e trans&ocation' @o/e$er, termina&&y

misfo&"e" protein cannot !e unfo&"e" for retrogra"e trans&ocation an" 8-D' @ence,termina&&y misfo&"e" protein or inso&u!&e protein aggregates cannot !e easi&y processe"

!y 8-D, an" autophagy may !e the on&y /ay to remo$e such protein aggregates'

• 8 stress !y &ipi" in?ury or cyto.ines an" su!seuent NB% acti$ation ha$e !een

imp&icate" as a cause of insu&in resistance'

• @igh g&ucose in"uces increase" insu&in synthesis /hich /i&& &ea" to an increase" amount

of unfo&"e" protein an" acti$ation of P8%, /hi&e &o/ g&ucose &ea"s to "ecrease"

synthesis of insu&in through eIC2 phosphory&ation' 8 stress or su!seuent NB%

acti$ation can &ea" to "ecrease" insu&in pro"uction, re"uce" ce&& mass or e$en ce&&

"eath

• -utophagy is important in the maintenance of appropriate P machinery an" autophagy

"eficiency cou&" &ea" to impaire" P response an" $u&nera!i&ity to/ar" 8 stress'

•

The mechanism of the compromise" P gene e#pression in autophagy"eficient ce&&sis not c&ear' e o!ser$e" impaire" e#pression of noncata&ytic regu&atory su!units of

PI%, p>5 an" p>5 (Euan et a&', 2)) that !in" to F!o# !in"ing protein ) (F=P) inan insu&in"epen"ent manner an" are important in the !asa& an" stimu&ate" e#pression of

"i$erse P genes (Par. et a&', 2)* innay et a&', 2)' Thus, "eficient p>5 an" p>5

e#pression might !e a cause of compromise" P in autophagy"eficient ce&&s'


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• The mechanism of 8 stress !y &ipi" in?ury or o!esity might entai& a&tere" 8 &umina&

homeostasis !y &ipi" o$er&oa", changes of &ipi" composition of 8 mem!rane, or

seuestration of 8 machinery to accommo"ate &ipi" "rop&ets' hen /e stu"ie" theimpact of &ipi"s on 8 in our system, /e confirme" that &ipi"s in"uce e#pression of

"i$erse P genes !oth in insu&inoma ce&&s an" primary is&et ce&&s, sho/ing that &ipi"s

are 8 stressors an" increase "eman" for P !y &ipi"s in those ce&&s'

• In"uction of P gene e#pression !y &ipi" /as significant&y &o/er in autophagy"eficient

ce&&s compare" to /i&"type ce&&s, suggesting that "eman" for P is increase" !y

&ipi" in?ury an" the increase" "eman" for P is unmet in autophagy"eficient ce&&s'

• -utophagy"eficient ce&&s /ere more suscepti!&e to in?ury !y high "oses of pa&mitic

aci", the most a!un"ant saturate" fatty aci" in $i$o' These resu&ts sho/ that autophagy"eficient ce&&s /ith compromise" P machinery are more $u&nera!&e to physio&ogica&

8 stressors as /e&& as pharmaco&ogica& 8 stressors'

• Increase" P gene e#pression of ce&&s of -tg7/i&" type (-tg7C0Co!0o! mice /asconfirme", suggests increase" "eman" for P !y o!esity in $i$o' Intriguing&y, -tg7Xce&&o!0o! mice "e$e&ope" se$ere "ia!etes, /hi&e &ittermate -tg7Xce&&o!0/ mice or

-tg7C0Co!0o! mice "e$e&ope" on&y mi&" hyperg&ycemia (Euan et a&', 2))'

• an"om !&oo" g&ucose &e$e& /as $ery high in -tg7Xce&&o!0o! mice, /hi&e it /as on&y

mi&"&y increase" in &ittermate -tg7Xce&&o!0/ or -tg7C0Co!0o! mice'

• -utophagy"eficient ce&&s are suscepti!&e to 8 stress impose" !y o!esity in $i$o

pro!a!&y "ue to compromise" P machinery' This hypothesis /as supporte" !y

increase" ce&& apoptosis an" mar.e" accumu&ation of reacti$e o#ygen species (1


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CP3Ao!0/ mice, /hich is apparent&y inconsistent /ith increase" autophagic

acti$ity'

• ysosoma& "egra"ation of &ong&i$e" proteins measure" !y counting re&ease of

incorporate" 3)4&eucine /as significant&y inhi!ite" !y pa&mitic aci" or o&eic aci" (P Y

')') (Euan et a&', 2)), suggesting that /hi&e &ipi"s enhance apparent autophagicacti$ity to e&iminate &ipi"s through Z&ipophagyZ (9572>2

• %inase acti$ity of PIB%) is essentia& for maintaining mitochon"ria& integrity an"

function in $i$o'

• PIB%) in"uces Par.in &oca&i+ation to the mitochon"ria in its .inase acti$ity"epen"ent

manner'

• The amount of hPar.in protein in the mitochon"ria& fraction /as high&y increase" upon

coe#pression /ith hPIB%) T' @o/e$er, hPIB%) .noc."o/n !y siB- "ecrease" the

amount of en"ogenous hPar.in in the mitochon"ria& fraction'

• The protein &e$e&s of se$era& mitochon"ria& proteins /ere not a&tere" !y changes in

PIB%) e#pression, in"icating that the change in Par.in &e$e&s in the mitochon"ria&

fraction is specifica&&y "ue to PIB%) &e$e&s'

• Par.in 32>< mutation "isrupts IB) "omain structure' The fu&& &ength of hPar.in /ith

32>< mutation, /hich is &oca&i+e" in aggresome&i.e structures, "i" not trans&ocate tothe mitochon"ria in the presence of PIB%) T'

• The intact IB) "omain of Par.in is reuire" for its trans&ocation to the mitochon"ria'

PIB%) promotes Par.in &oca&i+ation to the mitochon"ria !y regu&ating the &in.er region'

• coe#pression of hPar.in T)7502)7- mutant /ith hPIB%) mar.e"&y re"uce" the transfer

of the Par.in mutant to the mitochon"ria' hPar.in T)7502)78, a phosphomimetic mutant

!y mutating the threonine resi"ues to g&utamate, e#presse" it a&one an" foun" that it /as

most&y &oca&i+e" in the mitochon"ria' These resu&ts strong&y suggest that the phosphory&ation of hPar.in T)75 an" T2)7 resi"ues is critica& for its mitochon"ria&

&oca&i+ation'

• e generate" Btermina& truncate" hPIB%) constructs (XhPIB%) T an" %D, The

T)75containing hPar.in pepti"e, not the T2)7containing pepti"e, /as phosphory&ate"

!y XhPIB%) T, /hereas XhPIB%) %D sho/e" mar.e"&y re"uce" phosphory&ation of

the T)75containing pepti"e' PIB%) "irect&y regu&ates Par.in mitochon"ria& &oca&i+ationthrough T)75 phosphory&ation'


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PMID: 252)767

• Decrease" comp&e# I acti$ity, increase" mitochon"ria& DB- mutations, an" e#posure to

en$ironmenta& to#ins that inhi!it comp&e# I of the e&ectron transport chain ha$e a&& !een

associate" /ith PD'

• PIB%), a norma&&y short &i$e" mitochon"ria& protein .inase, nee"s to accumu&ate to high

&e$e&s on the outer surface of such "epo&ari+e" mitochon"ria in or"er to trigger Par.in

recruitment an" mitophagy'

• Par.in is autoinhi!ite" un"er !asa& con"itions, an" !ecomes "einhi!ite" in response to

mitochon"ria& "epo&ari+ation an" PIB%) .inase acti$ity' Par.in then u!iuitinates

mitochon"ria& proteins, such as the mitofusins an" $o&tage"epen"ent anion channe&s,/hich u&timate&y &ea"s to mitochon"ria& "egra"ation !y autophagy'

• The short mitochon"ria& isoform of -C (sm-C, an a&ternati$e trans&ation pro"uct of

the tumor suppressor p)9-C, &oca&i+es to mitochon"ria, /here it in"uces their "epo&ari+ation an" autophagy'

• 8#pression of sm-C in ce&& &ines an" in neurons "epo&ari+es mitochon"ria an" triggers

mitophagy in a Par.in an" PIB%)"epen"ent fashion, /ithout the reuirement of

chemica& uncoup&ers' The fin"ings p&ace sm-C upstream of PIB%) an" Par.in in a

no$e& signa&ing path/ay, /hich may she" &ight on the intrinsic mechanisms of PIB%)0Par.in mitophagy in PD'

• p)9-C, a tumor suppressor gene in the IB%40-C &ocus, enco"es p)9-C, a )69

amino aci" protein pre"ominant&y &oca&i+e" to the nuc&eus'


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• sm-C, an a&ternati$e trans&ation pro"uct of p)9-C mB-, /as "emonstrate" to

in"uce mitochon"ria& "epo&ari+ation an" autophagy in @8%29 ce&&s'

PMID: 252)767

•

Ea' ;- a&so p&ays other ro&es in antiapoptosis (in

et a&', 2)) an" chromosoma& sta!i&ity (Ohao et a&', 2)2'

•


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• hen ;P-' @o/e$er, this "e&ay /as not o!ser$e"

in


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• sing a 2pronge" approach, /e i"entifie" P8%@M) as an interaction partner of -=7

in its TP!oun" acti$e state, -=7 (TP' P8%@M) interacts "irect&y /ith a&&

M-P)30-=--P proteins through a high&y conser$e" 3interaction motif (I&ocate" !et/een the P&ec.strin homo&ogy "omain ) (P@) an" P@2 "omains of

P8%@M)'

• 8n"ogenous P8%@M) co&oca&i+es /ith -MP) at the cytoso&icfacing mem!rane, !ut

not the &umena& si"e, of 3=containing amphisomes0auto&ysosomes, in"icating that

P8%@M) is an autophagy a"aptor protein rather than a se&ecti$e cargo receptor'

• The he#americ @1P< comp&e# is an essentia& component of the &ate en"ocytic fusion

machinery an" is reuire" for auto&ysosome formation' P8%@M) interacts "irect&y /ith

the @1P< comp&e#, me"iate" !y the B "omain of P8%@M) an" the 3 terminus of ;P)45

• 3e&&u&ar mem!rane traffic.ing is regu&ate" !y sma&& TPases of the a!, -rf, an" -r&

fami&ies, /hich cyc&e !et/een inacti$e (DP an" acti$e (TP conformation states'

• TPases are anchore" to intrace&&u&ar $esic&es an" organe&&es, su!seuent&y regu&ating

recruitment of $arious effector proteins an" tethering factors, thus me"iating mem!ranefusion an" mo$ement of $esic&es'

• a!7 is an essentia& TPase that "efines the integrity of the &ate en"osoma&0&ysosoma&

compartment, regu&ating gro/thfactor receptor "egra"ation an" cationin"epen"entMannose6phosphate receptor (3IM6P retrie$a& to the transo&gi net/or., reuire"

for the sorting of aci"ic hy"ro&ases an" formation of functiona& &ysosomes'


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• Cusion of en"ocytic $esic&es is "ri$en !y tethering comp&e#es that !ring the $esic&es

together an" position them into c&ose pro#imity of the actua& fusion machinery' Tethering

comp&e#es, such as homotypic fusion an" $acuo&e protein sorting (@1P an" is therefore imp&icate" at ear&ier stages

of en"osoma& maturation, characteri+e" !y a!5 to a!7 con$ersion an" su!seuent

@1P< recruitment'

• P8%@M) interacte" "irect&y /ith @1P< comp&e# $ia its B "omain an" co&oca&i+e"

at $esic&e contact sites /ith a!7 an" @1P, !ut

not other u!iuitin&i.e proteins' pon seuence ana&ysis, /e "isco$ere" a potentia& 3

interaction region (I nest&e" !et/een the P&ec.strin homo&ogy (P@ "omains ) (P@)

an" 2 (P@2 of P8%@M)' 1n&y regions of

proteins through a high&y conser$e" type I motif'


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P&e.hm)\0\ M8Cs, "irect inhi!ition of mT1 /ith either %u6794 or Torin) resu&te"

in a mar.e" increase in 3=II an" p62 &e$e&s compare" to T M8Cs'

• co&oca&i+ation !et/een 3= an" -MP) /as significant&y re"uce" in P&e.hm)\0\

M8Cs' P&e.hm) is reuire" for the termina& maturation of autophagosome to

auto&ysosome, presuma!&y through faci&itating autophagosome&ysosome fusion'

• Dep&etion of P8%@M) resu&te" in the accumu&ation of CP in p620!positi$e

structures an" a 5L increase in processe" antigen presentation' This /as

significant&y enhance" upon the inhi!ition of autophagy !y M- !ut /as !&oc.e" !y proteasoma& inhi!ition' This effect /as compara!&e to si&encing of -T5 an" B=)'

• P8%@M) "ep&etion cause" the appearance of sma&& !positi$e (!A puncta in

untreate" ce&&s that /ere not present in contro& ce&&s' Moreo$er, /hi&e contro& ce&&s

efficient&y c&eare" puromycinin"uce" p62A0!A -I< aggregates, reco$ery of

P8%@M)"eficient ce&&s /as not apparent e$en after 6 hr, in"icating that P8%@M)

functions a&so "uring se&ecti$e, -I

Autophagy and Apoptosis

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