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Becausean enormous number ofdifferent combinations of these 20 modifications are possible, the proteinsbehavior can in principle be altered in a huge number of ways. Moreover, thepattern of modifications on a protein can determine its susceptibility to furthermodification, as illustrated by histone H3 in Figure 3-BlB.Cell biologists have only recently come to recognize that each protein's set ofcovalent modifications constitutes an importanl combinatorial regulatory code'As specific modi$ring groups are added to or removed from a protein, this codecauses a different set of protein behaviors-changing the activity or stability ofthe protein, its binding partners, and its specific location within the cell (Figure3-8iC).
This helps the cell respond rapidly and with great versatility to changesin its condition or environment.CellUnderliesA ComplexNetworkof ProteinInteractionsFunctionThere are many challengesfacing cell biologists in this "post-genome" era whencomplete genome sequences are knor.tm.One is the need to dissect and reconstruct each one of the thousands of protein machines that exist in an organismsuch as ourselves.
To understand these remarkable protein complexes, eachmust be reconstituted from its purified protein parts, so that we can study itsdetailed mode of operation under controlled conditions in a test tube, free fromFigure3-81 Multisiteproteinmodification and its effects.A proteinadditionthat carriesa post-translationalto morethan one of its aminoacidsideto carryachainscan be consideredregulatorycode.(A)Thecombinatorialoatternof known covalentmodificationsto the proteinp53;ubiquitinand SUMO(seeTable3-3).arerelatedpolypeptides(B)The possiblemodificationson the firstof20 aminoacidsat the N-terminushistoneH3,showingnot onlYtheirlocationsbut alsotheir activating(b/ue.)and inhibiting (red)effectson theadditionof neighboringcovalentmodifications.In additionto the effectsand methylationshown,the acetylationof a lysinearemutuallyexclusivereactions(seeFigure4-38).(C)Diagramshowingthe generalmannerin whichareaddedto (andmultisitemodificationsremovedfrom)a proteinthroughsignalingnetworks,and how theregulatorycoderesultingcombinatorialon the protein is readto alter its behaviorin the cell.188Chapter3: Proteinsall other cell components.
This alone is a massive task. But we now know thateach of these subcomponents of a cell also interacts with other sets of macromolecules, creating a large network of protein-protein and protein-nucleic acidinteractions throughout the cell. To understand the cell, therefore, we need toanalyzemost of these other interactions as well.We can gain some idea of the complexity of intracellular protein netvvorksfrom a particularly well-studied example described in Chapter 16: the manydozens of proteins that interact with the actin cytoskeleton in the yeast saccharomycescereuisiae(seeFigure l6-18).
The extent of such protein-protein interactions can also be estimated more generally. An enormous amount of valuableinformation is now freely available in protein databaseson the Internet: tens ofthousands of three-dimensional protein structures plus tens of millions of protein sequencesderived from the nucleotide sequencesofgenes. Scientistshavebeen developing new methods for mining this great resource to increase ourunderstanding of cells.
In particular, computer-based bioinformatics tools arebeing combined with robotics and microarray technologies (seep. s74) to allowthousands of proteins to be investigated in a single set of experiments. proteomics is a term that is often used to describe such research focused on thelarge-scaleanalysis of proteins, analogous to the term genomics describing theIarge-scaleanalysis of DNA sequencesand genes.Biologists use two different large-scalemethods to map the direct bindinginteractions between the many different proteins in a cell. The initial method ofchoice was based on genetics: through an ingenious technique known as theyeast two-hybrid screen (see Figure 8-24), tens of thousands of interactionsbetween thousands of proteins have been mapped in yeast,a nematode, and thefruit fly Drosophila. More recently, a biochemical method based on affinity tagging and mass spectroscopy has gained favor (discussedin chapter 8), becauseit appears to produce fewer spurious results.The results of these and other analyses that predict protein binding interactions have been tabulated and organized in Internet databases.This allows a cell biologist studying a small set ofproteins to readily discover which other proteins in the same cell are thought tobind to, and thus interact with, that set of proteins.
\Arhendisplayed graphicallyas a protein interaction map, eachprotein is representedby a box or dot in a twodimensional network, with a straight line connecting those proteins that havebeen found to bind to each other.\Mhen hundreds or thousands of proteins are displayed on the same map,the network diagram becomes bewilderingly complicated, serving to illustratehow much more we have to learn before we can claim to really understand thecell. Much more useful are small subsections of these maps, centered on a fewproteins of interest. Thus, Figure 3-82 shows a network of protein-protein interactions for the five proteins that form the SCFubiquitin ligase in a yeast cell (seeFigure 3-79). Four of the subunits of this ligase are located at the bottom right ofFigure 3-82.
The remaining subunit, the F-box protein that serves as its substrate-binding arm, appears as a set of 15 different gene products that bind toadaptor protein 2 (the Skpl protein). Along the top and left of the figure are setsof additional protein interactions marked with yellow and green shading: as indicated, these protein sets function at the origin of DNA replication, in cell cycleregulation, in methionine slmthesis, in the kinetochore, and in vacuolar H+ArPase assembly.we shall use this figure to explain how such protein interactionmaps are used, and what they do and do not mean.1. Protein interaction maps are useful for identifuing the likely function ofpreviously uncharacterized proteins. Examples are the products of thegenes that have thus far only been inferred to exist from the yeast genomesequence,which are the six proteins in the figure that lack a simple threeletter abbreviation (white lettersbeginning withy).
one, the product of socalled open readingframeYDRlg6c, is located in the origin of replicationgroup' and it is therefore likely to have a role in starting new replicationforks. The remaining five in this diagram are F-box proteins thai bind toSkpl; these are therefore likely to function as part of the ubiquitin ligase,serving as substrate-binding arms that recognize different target proteins.189P R O T E IFNU N C T I O NHowever, as we discussnext, neither assignment can be considered certainwithout additional data.2 .
Protein interaction networks need to be interpreted with caution because,as a result of evolution making efficient use of each organism's geneticinformation, the same protein can be used as part of two different proteincomplexes that have different types of functions. Thus, although protein Abinds to protein B and protein B binds to protein C, proteins A and C neednot function in the same process.For example, we know from detailed biochemical studies that the functions of Skpl in the kinetochore and in vacuolar H+-ATPaseassembly (yellow shading) are separate from its functionin the SCF ubiquitin ligase. In fact, only the remaining three functions ofsynthesis, cell cycle regulaSkpl illustrated in the diagram-methioninetion, and origin of replication (green shading)-involve ubiquitylation.3 .
In cross-speciescomparisons, those proteins displaying similar patternsof interactions in the two protein interaction maps are likely to have thesame function in the cell. Thus, as scientists generate more and morehighly detailed maps for multiple organisms, the results will becomeincreasingly useful for inferring protein function. These map comparisonsare a particularly powerful tool for deciphering the functions of humanproteins. There is a vast amount of direct information about protein function that can be obtained from genetic engineering, mutational, andO R I G I NO F R E P L I C A T I O NCELLCYCLEREGULATORSM E T H I O N I NSEY N T H E 5 I 5KINETOCHOREOkplE 2u b i q u i t i n coniugatingenzymeMit2ctfl9cCep3cbf2 .-..-Mcm2lMckl'/.adaptorprotein1VmTfpl'/'Ram2 -Vma2VACUOLARH*-ATPaseASSEMBLYadaptorprotein 2scaffoldprotein(cullin)Figure3-82 A map of some protein- protein interactionsof the SCFubiquitin ligaseand other proteins in the yeastS.lerevisiae,Thesymbolsand/or colorsusedfor the 5 proteinsof the ligasearethose in Figure3-79.
Note that 15 differentwith u/hitelettering(beginningwith Y) areonly knownfrom the genomeF-boxproteinsareshown(purpte);thoseof PeterBowersand DavidEisenberg,sequenceasopen readingframes.Foradditionaldetails,seetext.(CourtesyUCLA.)UCLA-DOEInstitutefor Genomicsand Proteomics,190Chapter3: ProteinsFigure3-83 A networkof protein-bindinginteractionsin a yeastcell.Eachlineconnectinga pairof dots (proteins)indicatesa protein-protein(FromA. Guimer6and M. Sales-Pardo,interaction.Mol.Syst.Biol.2:42,2006.With permissionfrom MacmillanPublishersLtd.)genetic analyses in model organisms-such as yeast, worms, and fliesthat is not available in humansThe available data suggestthat a typical protein in a human cell may interact with between 5 and 15 different partners. Often, each of the differentdomains in a multidomain protein binds to a different set of partners; in fact, wecan speculate that the unusually extensivemultidomain structures observed forhuman proteins may have evolved to facilitate these interactions.
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