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