Short Aggregation-Prone Peptide Detectives: Finding Proteins and Truths about Aggregation

Abstract

Inthe current issueof Journal of Molecular Biology,Ganesan et al. introduce a new and simple approachto specifically monitor the presence of proteinscontaining APSs (aggregation-prone sequences) [1].The authors use APS motif peptides to achievespecific binding to the target protein, and biotinylationof the peptide for sensitive detection in a Western-blot-type assay, which the authors designate as aPepBlot.The idea is interesting: β-aggregation is mediatedvia homotypic interactions of short motifs, and eachmoleculedonatesthesameinteractionstothefibril.Ina cross-β fibril, individual molecules come togetherin parallel or antiparallel β-sheets that stack via side-chain interactions, forming steric zippers [2].Whileseeding can effectively remove the lag time ofβ-aggregation, cross-seeding is remarkably ineffec-tive, demonstrating the preference for homotypicinteractions [3]. This result begs the question: is itpossible to exploit these homotypic interactions ofAPSs to specifically detect proteins? Ganesan et al.show support of this hypothesis for one Escherichiacoli aggregation-prone protein (β-galactosidase) andtwo human proteins (C-reactive protein and prostate-specific antigen) [1].The idea to take advantage of homotypic inter-actionsunderlyingβ-aggregationhaspreviouslybeenexploited in Grafted AMyloid-Motif AntiBODIES, orgammabodies, in which an aggregation-prone shortprotein sequence is grafted onto the complementar-ity-determiningregionofasingle-domainantibody[4].Gammabodies confer detection specificity and sensi-tivitycomparabletothatofregularantibodiesobtainedthrough immunization [4]. In the current report,Ganesan et al. show that it is possible to remove theantibody portion until only the aggregation-pronepeptide is left [1]. These probe peptides conferspecificity to the detection of the target protein incrudemixturessuchasbacteriallysateandbloodandseminal serum, and they have similar sensitivity toimmunization-derived antibodies [1].APS probe peptides may thus replace immuniza-tion-derived antibodies for the detection of aggre-gation-prone proteins in pathology applicationsand in research. Advantages include their modestprice, easy storage, and simple designability usingpredictorsofAPSs.Thepeptidesoffermotif-specificrecognition, whereas obtaining such antibodiesthrough traditional methods requires extensivescreening to distinguish them from antibodiesthat recognize composite epitopes, or from se-quence-independent, conformation-specific anti-bodies that recognize fibrils or toxic oligomers.Before the widespread use of the biotinylated probepeptidesinPepBlots,thefollowingfewopenquestionsremain to be explored:(1) Which protein conformation do the probepeptides recognize? Some gammabodies havebeen reported to bind to fibrils only, while othersrecognize monomers, β-sheet-rich oligomers, andfibrils [4], the three major species thought to beinvolved in β-aggregation (see Fig. 1A). The cause ofthesedifferencesbetweengammabodiesisunclearatthe moment but its elucidation will be necessary for abetter understanding of the PepBlot and hence thepossibilities and limitations of its use.(2) How accessible does the APS in the proteinhave to be? Many aggregation-prone proteins suchas α-synuclein, Aβ, and tau are largely intrinsicallydisordered, offering ready access to interactionpartners and probe peptides alike. On the otherhand, most folded proteins contain APSs that areusually prevented from aggregating by their inac-cessibility [5]. Even transient exposure ofthesemotifsthroughbreathingmotionsofthefoldedprotein core, by mildly denaturing conditions, or byshearforcesyieldsreadyaggregation;thisislikelythereason why most proteins can be induced to produce