Thou­sands on one chip: New Meth­od to study Proteins

Since the com­ple­tion of the human gen­ome an import­ant goal has been to elu­cid­ate the func­tion of the now known pro­teins: a new molecu­lar meth­od enables the invest­ig­a­tion of the func­tion for thou­sands of pro­teins in par­al­lel. Apply­ing this new meth­od, an inter­na­tion­al team of research­ers with lead­ing par­ti­cip­a­tion of the Tech­nic­al Uni­ver­sity of Munich (TUM) was able to identi­fy hun­dreds of pre­vi­ously unknown inter­ac­tions among proteins. The human gen­ome and those of most com­mon crops have been decoded for many years. Soon it will be pos­sible to sequence your per­son­al gen­ome for less than 1,000 Euros. At yet, there is a well-kept secret: for thou­sands of the roughly 20,000 – 30,000 pro­teins encoded in the gen­ome it is not clear what they do in the body, which func­tion they have. This makes it dif­fi­cult to inter­pret many upcom­ing data and under­stand the under­ly­ing molecu­lar pro­cesses – and this is the case in diverse fields such as med­ic­al research, plant research or the devel­op­ment of altern­at­ive energy sources. The func­tion of a pro­tein is a com­pos­ite of many dif­fer­ent aspects: with which pro­teins does it work togeth­er? How are its func­tions reg­u­lated and which pro­cesses are affected by it? Even for the ref­er­ence plant thale cress (Ara­bidop­sis thali­ana) the func­tion for about 10,000 pro­teins remains enig­mat­ic. Filling this know­ledge gap will take a long time using cur­rent meth­od­o­lo­gies. Elu­cid­at­ing these molecu­lar func­tions is there­fore of pree­m­in­ent import­ance.

Microar­rays enable the Invest­ig­a­tions of Thou­sands of Proteins

Pro­tein microar­rays allow the invest­ig­a­tion of thou­sands of pro­teins in a single exper­i­ment. Microar­rays are only a few cen­ti­meters in size and host thou­sands of indi­vidu­al test spots on very small space. To pro­duce stand­ard pro­tein microar­rays small amounts of pro­teins are prin­ted to a glass slide and chem­ic­ally fixed in each spot where they are then avail­able for exper­i­ments. How­ever, this approach requires the pri­or pro­duc­tion and puri­fic­a­tion of thou­sands of pro­teins, which is time con­sum­ing and expens­ive. Togeth­er these costs have pre­ven­ted the wide­spread use of pro­tein microar­rays des­pite their enorm­ous poten­tial. The research group of Pas­cal Fal­ter-Braun of the Chair of Plant Sys­tems Bio­logy at TUM togeth­er with col­leagues from the USA and Japan now achieved a pos­sibly decis­ive break­through: DNA, which is much easi­er and cheap­er to pro­duce, is prin­ted instead of pro­teins and the pro­tein arrays are sub­sequently ​‘developed’. DNA con­tains the inform­a­tion that spe­cifies the shape of pro­teins. After print­ing the DNA on the array the lat­ter is sub­merged in a reac­tion mix­ture that syn­thes­izes the pro­teins spe­cified by the prin­ted DNA. A chem­ic­al anchor that is attached to the glass sur­face rap­idly and tightly cap­tures the so developed pro­teins, which are then avail­able for func­tion­al stud­ies. The meth­od is called ​‘nuc­le­ic acid pro­gram­mable pro­tein array’ which, in con­junc­tion with the employed cap­ture agent, is abbre­vi­ated Halo-NAPPA. By using the new cap­ture chem­istry the research­ers were able to increase the dens­ity of the arrays such that it is now pos­sible to accom­mod­ate all pro­teins encoded in a gen­ome on just a few arrays. The sci­ent­ists could demon­strate the poten­tial of the pro­tein arrays in the con­text of plant hor­mone sig­nal­ing path­ways, which, for example, medi­ate responses to drought stress or against patho­gens.

1,000 nov­el Pro­tein-Pro­tein Inter­ac­tions discovered

For the study now pub­lished in PNAS inter­ac­tions of 38 of some of the most import­ant tran­scrip­tion factor pro­teins of thale cress were invest­ig­ated. Tran­scrip­tion factors determ­ine which genes are act­ive at what time and in which con­di­tions and con­sequently have a crit­ic­al role in organ­isms. The tran­scrip­tion factors them­selves can be activ­ated or inac­tiv­ated by inter­act­ing with oth­er pro­teins – in the present study nearly 1,000 new inter­ac­tions for the invest­ig­ated tran­scrip­tion factors were detec­ted using the pro­tein microar­rays. ​“Many of the now observed inter­ac­tions have nev­er been doc­u­mented. They will help us to under­stand how bio­lo­gic­al sys­tems and the under­ly­ing molecu­lar net­works func­tion”, says Fal­ter-Braun. Pro­teins in plants and in man do not act in isol­a­tion but have mutu­al reg­u­lat­ory rela­tion­ships and act togeth­er in com­plex net­works – the research focus of the TUM team around Fal­ter-Braun. In all organ­isms pro­teins have key roles and execute nearly all bio­lo­gic­al pro­cesses. ​“Pos­sibly, the new meth­od is a mile­stone towards under­stand­ing which pro­teins inter­act with which oth­er pro­teins or oth­er molecules in cells. Because it is cheap­er and sim­pler a wider range of research­ers can now work with these pro­tein arrays to invest­ig­ate pro­tein func­tions” says Fal­ter-Braun. The sci­ent­ist is con­vinced that the new meth­od will also help to accel­er­ate research in the research on renew­able ener­gies and the under­stand­ing of dis­eases.

Ori­gin­al publication

Jun­shi Yaza­ki, Mary Galli, Alice Y. Kim, Kazu­masa Nito, Fernando Ale­man, Kath­er­ine N. Chang, Anne-Rux­an­dra Carvunis, Rosa Quan, Hien Nguy­en, Liang Song, José M. Alvarez, Shao-shan Car­ol Huang, Huam­ing Chen, Niroshan Ramachandran, Stefan Alt­mann, Rodrigo A. Gutiér­rez, Dav­id E. Hille, Juli­an I. Schroeder, Joanne Chory, Joshua LaBaer, Marc Vid­al, Pas­cal Braun and Joseph R. Eck­er: Map­ping tran­scrip­tion factor inter­actome net­works using HaloT­ag pro­tein arrays, PNAS June 2016.
DOI: 10.1073/pnas.1603229113 Source: TUM