Micro Proteins

8/18/2019 Micro Proteins

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MicroProteinsSmall size-big impact

1

M. Raghavendra Date: 17.10.2015

2013BS09D Time: 3:00 PM

CreditSeminar


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MicroProteins (miPs)• Most biological processes require the formation of protein

complexes that are established through protein-proteininteraction (PPI) domains.

•

The formation stabilit! and disruption of protein complexes aredependent on "arious cellular factors such as regulator!

proteins and post-translational modifications.

• #ne important means b! $hich complex formation is regulatedis b! microProteins (miPs) mediated inhibition of complex

formation.

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Eguen et al. 2015


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MicroProteins (miPs)• MiPs are short usuall! single-domain proteins interact

(heterodimerize) $ith target and exert a dominant-negati"eeffect.

• %lthough some miPs are not small but the! are named so

-because the results of their actions are analogous to micro&'%s(mi&'%s) $hich are negati"e regulators of m&'%s.

• MiPs thus beha"e as post-translational regulators b! forminghomot!pic dimers $ith their targets and act through thedominantnegati"e suppression of protein complex function

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Eguen et al. 2015


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• The first miP identified is that disrupt a functional transcription factor

complexinhibitor of *'% binding (Id).• It $as isolated about t$o decades ago in a search for genes encoding basic helixloophelix (b+,+) transcription factors from a murineer!thro leuaemia cell c*'% librar!.

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Id protein

• Id is missing the basic region adacent to the +,+ domain that is essential forspecific *'% binding in another +,+ protein M!o*.

• In "itro studies sho$n that Id can bind $ith atleast three +,+ T/s (M!o*012 and 034)

• This interaction attenuate the abilit! to bind *'% as homodimeric orheterodimeric complexes 0x5 Id pre"ents M!o* from binding to *'%.

• &esearchers assumed that Id acts in a negati"e manner to fine-tune musclede"elopment.

• ,ater studies sho$ed that Id has a higher affinit! for the more ubiquitousl!expressed 0-t!pe b+,+ transcriptional regulators.

• Suggesting that Id probabl! regulates the abundance of M!o*60-t!peheterodimers and thus allo$s the M!o* homodimers to exert their action in atissue-specific manner.

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d: nhi!it"r "# D$% !inding!&'&: !a(i) he*i+ *"", he*i+


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Model of miP interference

-

• Targets of miPs are oftentranscriptional regulators that

bind to *'% as acti"ehomodimers.

•

miPs interfere $ith their targets b! forming non functional‑

heterodimeric complexes thatcannot bind to *'%.

DBD: D$%!inding d"mainPP: ,r"tein/,r"tein intera)ti"n d"main Staudt ene* 2011


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+omot!pic and heterot!pic miP inhibition

• MiPs can form inhibitor!complexes b! interacting $ith proteins that ha"e protein protein interaction (PPI)domains

• +omot!pic miP5 similar PPI (%)

• +eterot!pic miP5 non-identicalcompatible PPI (7)

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Eguen et al. 2015


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MiP( inhi!iti"n are re,"rted in,*ant(


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+,+ miP inhibition in plants• %rabidopsis ( Arabidopsis thaliana) brassinosteroid-sensiti"e +,+

P%8,#79T&%:#, &0SIST%'T1 (P&01; 11 *a) regulates petiole length.

• In rice (Oryza sativa) homolog I'8&0%S0* ,%MI'% I'8,I'%TI#'1(I,I1; 11 *a) lamina oint inclination (an erect leaf).

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Staudt ene* 2011


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P&01 and I7+1 antagonisticall! regulate cell elongation

• #"erexpression of P&01 and I,I1 increased7&-induced cell elongation in both%rabidopsis and rice.

• #"erexpression of I,1 7I'*I'< b+,+PT0I'1 (I7+1) leads to d$arfism in%rabidopsis and this phenot!pe is suppressed

b! o"erexpression of P&01 indicating thatP&01 promotes cell elongation b! inacti"atingI7+1.

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PRE1(u,,re((e( theee)t( "# B&1

"*: %ra!id",(i( tha*iana"*um!ia 6i*dt,e )"ntr"*.B&18+: vere+,re((i"n "# B&1PRE18+: vere+,re((i"n "# PRE1

Bai et al. 2012


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The basic domain is not required for I7+1 function

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• ari"u( degree( "# d6ar# ,hen"t,e "!(erved am"ng T1 ,*ant(

"vere+,re((ing 6i*d8t,e IBH1.• $um!er and ,er)entage "# tran(geni) ,*ant( (h"6ing ea)h

)ateg"r "# ,hen"t,e (everit "!(erved am"ng ,",u*ati"n( "#tran(geni) ,*ant( "vere+,re((ing 6i*d8t,e IBH1 "r mutant B&1)"ntaining de*eti"n "# the !a(i) d"main ;IBH1ΔB< "r "# the %Sd"main ;IBH1ΔAS


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I7+1 Interacts $ith +7I1 in vitro and in vivo• 7! %rabidopsis interactome database It $as found that I7+1 interact $ith b+,+ proteins 7002 %t2g1=>?? (+7I1 ) 7P08I71 %t@g3=@A?

• 7! !east t$o-h!brid assa!s it is confirmed the interactions.• 7oth +7I1 and 7002 interact $ith I7+1 in !east and this interaction needs the I7+1 %S

domain but not the basic domain.

• I7+1 interacts $ith P&01 through its +,+ domain and does not require the %S domain or basic

domain

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&B1:8 &M'= >BEE2 $TER%T$= T&

B&1.

BEE2: BR E$&%$EDE?PRESS$2

B1: @T&RME$TER%T$= B%S &'&

Bai et al. 2012


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+7I1 promotes cell elongation

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• Re,re(entative ,*ant( "# the 6i*d t,e and HBI1 )"8(u,,re((i"n ;S< and "vere+,re((i"n ;A1 and 2A<*ine( 6ere gr"6n in ("i* #"r 4 6ee(.

• uantitative RT8PR ana*(e( "# HBI1 e+,re((i"n in,*ant(

Chang et al. 2009


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I7+1 and P&01 antagonisticall! regulate the acti"it! of +7I1

• #"erexpression of IBH1 or nocdo$n the expression of PRE ssuppresses the phenot!pe of HBI1-Ox. Plants and detached lea"es.

14Bai et al. 2012


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P&0-I7+1-+7I1 regulator! chain controls cell

elongation• #"erexpression of P&01 and I,I1 leads to increased petiole length

and lamina oint inclination in %rabidopsis and rice respecti"el!and increased sensiti"it! to brassinosteroids (7&).

• 8on"ersel! I7+1 o"erexpression leads to reduced sensiti"it! to 7&

smaller petioles in %rabidopsis and reduced lamina inclination inrice

• #"erexpression of +7I1 results in longer petioles and longerh!pocot!ls in %rabidopsis.

• The antagonistic effect of miPs on their target proteins is a commontrait in miPs.

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&>-MB7 miP

1-


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Single repeat &>-MB7 transcription factor

• Sequence signature C*60D,x2C&6EDx>,xA,x>& required for the interaction bet$een

&> MB7s and &67-lie b+,+ transcription factors.

• &> MB7s contain a sequence motif FxM required for its cell-to-cell mo"ement

• &> MB7s follo$ a pattern of Mx1=Fx1=F instead of C/6IDx1=Fx1=F that is t!picall!

found in the &> MB7 repeat of &2&>-MB7 proteins and MB7,2.

• % single MB7 repeat &> MB7s lac the acti"ation domain that is t!picall! presentin most transcription factors

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1. TRPT@&$;TR@<

2. %PRE ;P<3. E$&%$ER >

TR@ %$D P1;ET1<

4. ET25. ET3%PRE8

'E M@B3;P'3<

-. TR&ME'ESS1;T'1<

7. T'2P'4

ang hen 2014


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• The fe$ functions of &>-MB7 are• Trichome de"elopment &oot hair de"elopment anthoc!anin

accumulation and stomatal formation.

• The etc3cpl3 mutant $as sho$n to flo$er earlier $ith fe$er lea"es thanthe $ild-t!pe.

•

0 and TT


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Promoter acti"it!• The differences in tissue6organ expression patterns of &> MB7sdetermine the first le"el of specificities.

• %mong all se"en &> MB7 genes ET"# and T"$1 T"$#"P$% $as also notdetected or at "er! lo$ le"el in the root . %ll of these three genes tested $ereable to partiall! rescue the root hair phenot!pe of cpc mutant $hen their

expression $as dri"en b! "P" promoter.• &egulation of expression of &> MB7 genes is important for their

functioning .• The constructs ( pET"3&ET"3 pTR!&ET"3 and p"P"&ET"3) $ere used to

complement triple mutants containing etc3'• pET"3&ET"3 construct rescued the cpc try etc3 mutant to the same extent

• pTR!&ET"3and p"P"&ET"3 displa!ed an o"er-rescued phenot!pe resembling that of try.

• Promoters of &>-MB7s are interchangeable (single mutant rescue)• Fhen ET"3 $as expressed under the promoter of ET"3 TR! or "P" it could

equall! rescue the trichome phenot!pe of etc3 mutant.

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/unction of &>-MB7s in trichome de"elopment• Trichomes are hair cells produced b!the out$ard gro$th of epidermal cells.

• Trichome initiation is promoted b! anacti"ator complex consisting of aF*3?-repeat protein

T&%'SP%&0'T T0ST% MB7-t!petranscription factor ig: R3 MYB transcription factors function redundantly to control trichomeformation in Arabidopsis. n "* 6i*d t,e ,*ant ;*e#t


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Modes of action of &>-MB7s

• TT MB7 genes.

•


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,ittle Zipper proteins (:P&)

are miPs

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:P& proteins are structurall! similar to :IP motif

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•

Ph!logenetic anal!sis re"ealed that the! can be classified into t$odistinct groups5

1. :P&1 and :P&2

2. :P&> and :P&3

• :P&1 and :P&2 ha"e '-terminal extensions $hich pla! a role in *'% binding.

CP m"ti# i( *")ated1. 8termina* regi"n( "# CPR1

and CPR2.2. $8termina* regi"n( "# CPR3

and CPR4.

• CPR Pr"tein )"ntain a (ing*e *eu)ine Ii,,er ;CP< d"main thatena!*e( them t" !ind and regu*ate )*a(( &"me"d"main.

• CPR3 and CPR4 ,*a r"*e in S%M Deve*",mentF CPR1 and CPR2*ined t" ,atterning "# *ea# ,"*arit

ene* et al.

2007


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&egulator! modules ,ITT,0 :IPP0& (:P&s)

• :P& Protein bind and inhibit class III +omeodomain-leucine zipper(+*-:IPIII) proteins acti"ation.

• +* :IP III are conser"ed plant proteins that act as potent regulatorsof ad6abaxial polarit! in Arabidosis'

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>ig: CPR ,r"tein( inhi!it &D8CPD$% !inding a)tivit ! #"rming

heter"dimer( 6ith them

im et al. 200


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MI'I :I'8 /I'


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MI'I :I'8 /I'


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Protein structure of MI/ and zinc finger

• The MI/1 gene encodes protein of 1?1aminoacid residues.

• It is similar to '-terminal region of:/-+* proteins

• The putati"e :/ domain contains eightc!steine and6or histidine residues(CG>HG11CG12(2ACG2CGCHG>H)

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>u** *ength amin"a)id (eJuen)e"# M>1

&u Ma 200-


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0xpression pattern of MI/ genes in Arabidopsis

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% re,re(entativee+am,*e "# three RT8

PR e+aminati"n( "#MIF1F MIF2F and MIF3K APT1 i( u(ed a( theinterna* )"ntr"*K the))*e num!er( "# thePR rea)ti"n areindi)ated at the right"# the image(.&u Ma 200-


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Phenot!pic reco"er! of transgenic plants o"erexpressing :/-+*@ b!

MI/1 co expression

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• The MI/1 or :/-+*@ geneo"erexpressed under controlof 8aMH >@ S promotor.

• 0xpression of MI/1 and :/-+*@ genes in transgenic

plants (transcript le"els ofq&T-P8&).

&"ng et al. 2011


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MI/1 act as dominant negati"e regulators of :/-+*@ transcription factors

• Working model ofMIF1. The MI/1 proteininhibits nuclearlocalization of the :+*@

protein b! formingMI/1-:+*@heterodimers in thec!toplasm. In thenucleus the MI/1-:+*@heterodimers ha"e a

lo$er *'% bindingacti"it! compared $iththat of the :+*@homodimers.

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There are 2? published bona,ide %rabidopsis miP

31Eguen et al.


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Fhat is not miP

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Proteins $ith sequence similarit! to T/s but lac a *7*

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• The auth"r( identiLed 438 T>8re*ated ,r"tein( and re#erred t" the(e a(,utative miP(.

• The a!(en)e "# a DBD d"main d"e( n"t ne)e((ari* *ead t" aninhi!it"r ee)t

• But the(e d"e( n"t Lt int" deLned miPF (u)h a( (iIe and d"minant8negative m"de "# a)ti"n.


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,ITT,0 SIPP0& (SPP&)

• @2-*a multidomain protein thatregulates +*-:ip IH /,#F0&I'<F%


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Thus it $as concluded that SPP& is a miP that functions s!nergisticall!$ith its target; ho$e"er most no$n miPs are no$n for their antagonisticeffect on $ild t!pe alleles.

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Testin the e!ect of S""R on#$A•P*ant( )arr the fwa-1 mutati"n(h"6 a *ate G"6ering ,hen"t,e.•vere+,re((ed SPPR u(ing)"n(titutive 35S ,r"m"ter in 6i*d8t,e "r fwa-1 !a)gr"und(•Seven "# eight inde,endenttran(geni) *ine( "vere+,re((ingSPPR in the fwa-1 !a)gr"undG"6ered (igniL)ant* *aterthan fwa-1 ,*ant(.

Mangnani et al.2014


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&0SP#'S0 T# %7S8ISI8 %8I* %'* S%,T1 (&%S1)

• &%S1 (2A*a) predicted to be in"ol"ed in salt response related to

T


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• The putati"e list of 3=> potential miPs that $ere published ranged insize from > to 12@ *a.

• %pproximatel! 4?K of $hich $ere abo"e 2?*a size.

•

This list contains some no$n miPs and probabl! some undisco"eredmiPs.

• Most of the proteins in list are better classified as modulator! proteinsdue to their size

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MiP biogenesis

3


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+o$ did miPs arise

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M"(t miP( are trans8miP(

Mangnani et al.2014


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8is miPs

• M%*S box box T/ genes /,#F0&I'< ,#89S M (/,M) andS+#&T H0


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:P& genes are ph!logeneticall! deri"ed from the 8>+*: genes.

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• CPR ev"*uti"n: degenerati"n "# an an)ient 3&DC du,*i)ate gene;eu,h**",hte: #ern(


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,iterature cited

• 7ai M. /an M. #h 0. N Fang :. (2?12) % triple helix-loop-helix6 basic helix-loop-helix cascade controls cell elongation do$nstream of multiple hormonal anden"ironmental signalling path$a!s in %rabidopsis. The Plant "ell 24 3O14-3O2O.

• 0guen T. Straub *.


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• +u F. N Ma +. (2??A) 8haracterisation of a no"el putati"e zinc finger

gene MI/15 in"ol"ement in multiple hormonal regulation of %rabidopsisde"elopment. The Plant 0o.rnal 4 >OO-322.

• Eim B. Eim S. ,ee M. ,ee I. Par +. Seo P. ung . E$on 0.Suh S. F. Pae E. N Par 8. (2??=) +*-:IP III acti"it! is modulated

b! competiti"e inhibitors "ia a feedbac loop in %rabidopsis shoot apicalmeristem de"elopment. The Plant "ell 20 O2?-O>>.

• Mangnani 0. Elein '. 'am +. Eim . Pham E. /iume 0.Mudgett M.7. N &hee S.B. (2?13) % comprehensi"e anal!sis ofmicroproteins re"eals their potentiall! $idespread mechanism oftranscriptional regulation. Plant Physiolo/y) 16 13O-1@O.

• Pose *. Herhage ,. #tt /. Bant ,. Mathieu . %ngenent


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• Staudt %. N Fenel S.(2?11) &egulation of protein function b! QmicroproteinsR. E*BO Reports 12 >@-32.

•

Fang S. N 8hen . (2?13) &egulation of cell fate determination b! single repeat&>-MB7 transcription factors in %rabidopsis. +rontiers in Plant cience (1>>)1-11.

• Fang S. E$a S. :eng J. 0llis 7.0. 8hen G. Schiefelben . N 8hen .(2??4) T&I8+#M0,0SS1 regulates trichome patterning b! suppressing=4>->==2.

•

Fenel S. 0mer! . +ou 7. 0"ans M. M. S. N 7arton M.E. (2??4) % feedbacregulator! module formed b! ,ITT,0 :IPP0& and +*-:IPIII genes. The Plant"ell 1! >>4O->>O?.

• :hang ,. 7ai M. Fu . :hu . Fang +. :hang :. Fang F. Sun B. :hao. Sun G. Bang +. Gu B. Eim S. /uioa S. ,in F. 8hong E. ,u T. NFang :. (2??O) %ntagonistic +,+6b+,+ transcription factors mediate brassinosteroid regulation of cell elongation and plant de"elopment in &ice and%rabidopsis. The Plant "ell 21 >4A4->4=?.

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8#'8,9SI#'

• Microproteins (miPs) are small proteins that disrupt functional complexes.

• MiPs ha"e a dominant-negati"e mode of action.

• %ll no$n bona ,ide Arabidopsis miPs are less than 2? *a.

• Transcription factor-lie proteins lacing dominant-negati"e effects are

not mips.

• Microproteins (miPs) are short usuall! single-domain proteins that inanalog! to mi&'%s heterodimerize $ith their targets and exert adominant-negati"e effect.

• Modulator! proteins misrepresented as miPs do not qualif! as true miPs

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Micro Proteins