Dr B.R.

Ambedkar Center Ior Biomedical Research

University oI Delhi
Pioteins can moonlight!!
WhaL ls MoonllghLlng ??
O he iuea of one gene-one piotein-one function has become
too olu.
"Moonligbting-0lu pioteins leaining new tiicks"
O ncieasing numbei of pioteins aie founu to peifoim two oi
moie functions
nsight of Noonlighting..
O Buman genome encoue few pioteins, than expecteu fiom
such a complex oiganism having a laige genome.
O 0ne gene may encoue foi pioteins with moie than one
function.
O vaiious ways
Alteinative splicing
BNA ieaiiangement
Post tianslational mouifications of pioteins
uene Buplication with uiffeiential mutation
uene Fusion
nsight of Noonlighting..
^otein moonligbting oi gene sba^ing) is a phenomenon
by which a piotein can peifoim moie than one function.
Excluding-
W plice vaiiants
W uene fusions
W Bomologous but non iuentical piotein
W Post tianslational mouifications
W ame function -Biffeient cellulai location oi substiates
veiy special multifunctional pioteins,
Nultiple autonomous, often unielateu, functions without
paititioning these functions into uiffeient piotein uomain
pleiotiopism
ingle gene-
multiple
pathways
Nultiple
phenotypic
tiaits
nactivation
Piotein
moonlighting
ingle gene-
piotein with
multiple functions
ingle function
affecteu
Nutation in
couing iegion
MoonllghLlng and leloLroplsm
lscovery
iystallins
metabolism
Buck-lactate
uehyuiogenase
uitle-enolase
Eye
tiuctuial
iolevision
Pioteins that can moonlight
O Enzymes
O Receptois
O on channels
O hapeiones.
'he most common piimaiy function of moonlighting
pioteins is enzymatic catalysis, but these enzymes have
acquiieu seconuaiy non-enzymatic ioles.'
moonlighting functions seconuaiy to catalysis incluue
signal tiansuuction, tiansciiptional iegulation, apoptosis,
motility, anu stiuctuial functions.
Examples of moonlighting-pioteins fiom uiffeient kinguoms

MoonllghLlng roLelns Molecular Mechanlsms

O Biffeiential localization
O Biffeiential expiession
O Liganuubstiate concentiation
O Binuing sites
O omplex foimation
O 0ligomeiization
Noleculai mechanisms of Noonlighting
lfferenLlal locallzaLlon
he Fscbericbio coli PutA protein
ln Plosmo membrone
pioline uehyuiogenase anu pyiioline-- caiboxylate
uehyuiogenase activity when associateu with PN.
ln Cytoplosm-
lacks enzymatic activity anu binus BNA as a tiansciiptional
iepiessoi of put genes.
nslde and ouLslde Lhe cell
O Pbospboqlucose isomerose
atalyzes the seconu step in glycolysis, the intei-conveision
of glucose -phosphate anu fiuctose -phosphate .
O ecieteu by cells anu plays at least auuitional ioles.
Neuioleukin - both..
a cytokine - causes B cells to matuie into antibouy-secieting
cells,
a nerve qrowtb foctor that piomotes the suivival of some
embiyonic spinal neuions anu some sensoiy neives.
Autociine Notility Factoi ANF) -
a cytokine that stimulates cell migiation .
A Biffeientiation anu Natuiation Neuiatoi BNN) -
cause Vifferentiotion of human myeloiu leukemia cells
lfferenLlal Lxpresslon
O europilin -
ell uiface Receptoi on enuothelial cells,
-uetects vasculai enuothelial giowth factoi anu inuicates
when new bloou cells aie neeueu.
ell uiface Receptoi in neive axons,
-uetects a uiffeient liganu, emaphoiin , anu helps steei
axons to theii piopei uestinations.
llgomerlzaLlon
O lycerolVebyVe--pbospbote VebyVroqenose
tetiamei,
conveits glyceialuehyue--phosphate to
, uiphosphoglyceiate.
monomei,
it is a nucleai 0iacil-BNA glycosylase-
iemoving uiacil that is piesent in BNA because of
acciuental use of u0P uuiing BNA synthesis oi
ueamination of cytosine iesiuues.
omplex lormaLlon
O F. coli tbioreVoxin
ueoxyiibonucleotiue synthesis: it helps to ieuuce
iibonucleosiue uiphosphates to ueoxyiibonucleosiue
uiphosphates.
ieciuiteu by phage, in which it functions as a subunit of
a heteiouimeiic BNA polymeiase.
O PutA
O Aconitase
Fe-uepenuent enzyme that has catalytic activity only when
cellulai iion concentiations aie high.
4 When the iion concentiation uecieases - ion Responsive
Element Binuing Piotein RE-BP).
4 By binuing to a stem-loop, the RE, in the '-untianslateu
iegion of feiiitin mRNA, the RE-BP pievents the synthesis of
feiiitin.
4 REs in the '-untianslateu iegion of the tiansfeiiin ieceptoi
mRNA anu contiols the uegiauation of tiansfeiiin ieceptoi
mRNA.
4 not both simultaneously.
lgand /SubsLraLe oncenLraLlon
lndlng SlLe
O he F. coli aspaitate ieceptoi, which functions in
bacteiial chemotaxis, is also a Naltose Binuing Piotein
NBP) Receptoi.
uiffeient but oveilapping binuing sites foi aspaitate anu NBP
O Aconitase active site is the suiface useu foi RNA
binuing when the piotein functions as the RE-BP.
ecenLly rysLalllzed MoonllghL roLelnIAn|I maturase
O roup l intron splicinq foctor anu a bominq enVonucleose
he piotein is encoueu in an intion of the Asperqillus
niVulons mitochonuiial apo-cytochiome b gene.
O homing enuonuclease-
-Ani initiates tiansfei of the intion by cleaving a BNA
taiget sequence within a homologous allele that lacks the
intion sequence.
O plicing of the intion RNA uuiing post-tiansciiptional
excision fiom piecuisoi RNA.
. A Resolution X-iay ciystal stiuctuie of the -Ani
matuiase in complex with a base paii uuplex BNA
fiagment containing the native homing site
-Ani matu^ase..
IAn|I maturase
O Although the ciystal stiuctuie uoes not incluue RNA, seveial
pieces of eviuence inuicate that the suiface of -Ani involveu
in RNA binuing is uistinct fiom the BNA-binuing sites.
O wo mutations that pieventeu BNA binuing anu cleavage uiu
not affect RNA matuiation activity
IAn|I maturase
O Fuithei stuuies iuentifieu clusteis of basic amino acius in the N-
anu -teiminal uomains.
O uistant fiomthe BNA binuing sites.
O A potential suiface foi inteiacting with the RNA
Aig
ulu
ild-type -Ani
Mutant -Ani
9
enfold slowe^ RNA splicing and lowe^ affinity fo^ RNA
MuLaLlonal analysls
IAn|I maturase
O iystal tiuctuie Anu Nutational Analysis-
stiongly suppoit the mouel of two inuepenuent functional
suifaces foi the enVonucleose anu moturose activities.
goou example of a piotein in which a seconu activity, the
matuiase activity, evolveu fiom changes in an unuseu
solvent exposeu suiface aiea
ow does one ldenLlfy roLelnMoonllghLlng ??
O 0sually Ty cbance fiom activity stuuies
O mmuno bistocbemist^y oi Mass Spect^omet^y to
uetect theii sub-cellulai location, tissues, cell-types.
O By mutational analysis
denLlflcaLlon
eterminotion by Structure Ieotures-
O -^ay C^ystallog^apby, NMR, Laige size of piotein anu
unuseu suiface aiea may suggest foi its multiple function.
O ystem Biology applications like nte^actomics, to know with
what it inteiacts.
O ompaiison with oithologous pioteins with the same
piimaiy function.
O With time, uevelopment of statistical, uata-mining anu
machine leaining appioaches will help to examine these
pioteins anu making pieuictions.
LvoluLlon
O Noonlighting Pioteins- ueneially Bighly onseiveu,
"Why moonlighting functions aie so fiequently iuentifieu in
highly conseiveu pioteins."
O 0biquitous in all kinguoms, has been aiounu foi ovei a billion
yeais of time - ample time foi evolution
O Example- ugaiglycolytic) pathway enzymes- out of aie
moonlighting enzymes.
O Noonlighting functions also seem to occui moie often in
pioteins that aie constitutively expiesseu at ielatively high
levels
Why Noonlighting-Pioteins evei came into being.
O o expanu the functional capabilities of an oiganism without
the buiuen of an expanuing genome size . contiauicts the
piesence of laige amount of Non-ouing BNA)
O inkeieis way of Evolution -
which means that theie is no enu goal in evolution anu that
novel functions only uevelop by auapting existing ones.
f a paiticulai novel function iesults in an auvantage foi the
oiganism, this function will be selecteu uuiing evolution.
tiuctuial Featuieshanges in Noonlighting-Pioteins
O ome enzymes aie laigei than necessity foi theii function,
unuseu solvent exposeu suiface anu many pockets on the
piotein suiface coulu be mouifieu to make the auuitional
binuing sites.
O New use of existing binuing sites oi mouifications of the
unuseu iegions.
O Low specificity of the active site-mutations in active site oi
use of uiffeient iegions suiiounuing it.
Benefits
O Piokaiyotes - Fewei pioteins to synthesize, less BNA to ieplicate-
Soves Fnerqy
O CoorVinotinq Cellulor octivities- ome pioteins with one activity
iegulate othei pioteins with similai activity-Regulation of
Epithelial ouium on hannel by FR hannel-maintains
epithelial cell homoeostasis.
O Self requlotion of tronscription onV tronslotion- Nany biosynthetic
oi catabolic enzymes iegulate theii own synthesis
hymiuylate ynthase- binus to stem loop stiuctuie at '-enu of
mRNA
Put A piotein
eneflLs
O Fffective cellulor response- hiombin that tiiggei multiple
pathways
O Switcbes between potbwoys- seveial pioteins aie both
pioteases anu chapeione activity- FtsB, yeast mitochonuiial
homolog of FtsB-AfgP, RcaP)
O oiielation between multiple functions - yeast FtsB
O ometimes no cleai connection- banu piotein in RB's
plasma membiane
Neuical Relevance
O he complex phenotypes of seveial uisoiueis, may be ielateu
to the involvement of moonlighting pioteins.
O ibyVrolipoomiVe ebyVroqenose { -
Nitochonuiial Enzyme
component of at least uiffeient multi-enzyme complexes
ciitical foi eneigy metabolism anu ieuox balance
ueficiencies in BLB activity aie associateu with seveie
uisoiueis in infancy, such as an inability to thiive,
hypotonia anu metabolic uisoiueis.
O viiulence piopeities, paiticulaily invasion
O ycobocteriumtuberculosis glutamate iacemase Nui )
cell wall peptiuoglycan) biosynthesis
O Nui -BNA gyiase inhibitoi, by ieuucing binuing of gyiase to BNA
O Pievents action of the cipiofloxacin because Nui inhibits binuing of
gyiase to BNA
O ytotoxic uouble-stianu BNA bieaks x
O BNA ieplication inhibition X
Noonlighting-Pioteins in Bacteiial Woilu..
MoonllghLlng AcLlons of acLerlal MeLabollc roLelns
MoonllghLlng AcLlons of acLerlal MeLabollc roLelns
oncludlng wlLh Lasks ahead
O heie is a gieat ueal of inteiaction between maciomolecules,
anu the mouein cell is a sophisticateu anu highly oiganizeu
netwoik.
O t auus to the uifficulty of inteipieting genome sequences.
O Nany pioteins can have multiple inteiactions, it coulu be
uifficult to asceitain who inteiacts with whom in the cell.
"Noonlighting functions cieate a whole new level of
complexity in the cell. A moonlighting piotein may
link a metabolic pathway to a signalling pathway in a
completely unexpecteu mannei"
eferences
O effeiy anuaiy 999). Noonlighting pioteins. renVs Biocbem. Sci.
O effeiy Becembei ). Noleculai mechanisms foi multitasking:
iecent ciystal stiuctuies of moonlighting pioteins. Curr. 0pin. Struct. Biol.
O Bubeits BB, van uei Klei Apiil ). Noonlighting pioteins: an
intiiguing moue of multitasking" ournol of Biocbemistry onV Biopbysics
O Boluuc N, piegel P, hatteijee P, Biauy KL, Bowning NE, apiaia N,
Waiing RB, touuaiu BL: tiuctuial anu biochemical analysis of BNA anu
RNA binuing by a bifunctional homing enuonuclease anu gioup intion
splicing factoi. enes ev