304 CHIMIA 2018, 72, No. 5 News from New Chemistry Professors iN switzerlaNd doi:10.2533/chimia.2018.304 Chimia 72 (2018) 304–308 © Swiss Chemical Society Engineering Aspects of Protein Interactions and Self-assembly Miriam Linsenmeier and Paolo Arosio* Abstract: In the new Laboratory for Biochemical Engineering (LBCE) at ETH Zurich researchers combine prin- ciples of chemical engineering with microfluidic technology and biophysical methods to investigate the physical determinants of biomolecular self-assembly in living organisms. In this account, we show the impact of this activity on concrete applications in biomedical sciences and biotechnology. We focus in particular on the field of protein aggregation and phase separation, and we highlight examples in the context of diagnosis and treatment of Alzheimer’s disease and neurodegenerative disorders, cell compartmentalization as well as manufacturing and delivery of therapeutic proteins. Keywords: Alzheimer’s disease · Amyloids · Microfluidics · Protein aggregation · Protein interactions · Protein phase separation 1. Protein Structure and changes and even to single point mutations Interactions of the protein sequence, and is not free of mistakes. In some cases, aberrant interac- Self-organization is one of the funda- tions leading to misfolding and undesired mental processes underlying the origin aggregation can occur.[1] This is the case of life. The series of cellular functions on for instance of amyloids, which represent which life depends originate at the molec- a particularly important class of aberrant ular level from interactions between bio- aggregates of peptides and proteins which molecules, namely proteins and nucleic are involved in several human disorders acids. Proteins represent a unique class of including Alzheimer’s, Parkinson’s and lyophilic colloids with heterogeneous and systemic amyloidoses.[2] Amyloids can Paolo Arosio obtained his master’s complex structures, which exhibit ampho- originate from a broad range of very dif- degree in chemical engineering from the teric polyelectrolyte nature and the simul- ferent peptides and proteins and exhibit Politecnico di Milano in 2007, and his taneous presence of hydrophobic and hy- a remarkably consistent supramolecular doctoral degree from the Swiss Federal drophilic patches. Proteins are synthesized structure, which is mostly independent Institute of Technology ETH Zurich in as linear chains of amino acids which can of the precursor protein and consists of a 2011, working under the supervision of fold into suitable secondary and tertiary diameter of few nanometers, a length of Prof. M. Morbidelli. His PhD work was 3-dimensional structures. In some cases, several microns, and a predominant cross awarded the ETH Medal. Subsequently the stability and the activity of proteins β-sheet secondary structure.[2c] This com- he carried out postdoctoral research at the require the formation of quaternary struc- mon structure is primarily due to the high Department of Chemistry at the University tures, such as complexation into oligomers number of hydrogen bonds that proteins of Cambridge, UK, with Prof. T. P. J. or assembly into large filaments. can form along the β-sheet structure. In Knowles, funded by the Swiss National Structure and intermolecular forces addition to these aspecific interactions, the Science Foundation (SNSF) and by the are two strongly interconnected aspects in thermodynamic stability of the fibrils is European Marie Curie Fellowship scheme proteins: evolution has optimized the for- further promoted by the exclusion of water for career development. In 2016 he re- mation of secondary, tertiary and quater- molecules and specific side-chain interac- turned to ETH as Assistant Professor in nary structure by controlling a delicate bal- tions within the inner core of the fibrils.[2c] Biochemical Engineering. His research ance of intramolecular and intermolecular Although amyloid structures were interests focus on understanding and con- forces, which include electrostatic, van originally discovered in association with trolling protein self-assembly and protein der Waals, solvent and hydrophobic inter- a variety of pathological conditions,[2a,c] interaction processes that underlie prob- actions. In particular, hydrophobic forces increasing evidence demonstrates that am- lems of key fundamental and practical im- play a major role in protein folding, since yloids underlie a series of physiological portance in biology and biotechnology. globular proteins show stable native struc- functions,[3] including the natural storage tures in which hydrophilic groups are sol- of peptide hormones,[4] the formation of vent-exposed, while hydrophobic patches adhesive microbe biofilms[5] and cell com- are buried intramolecularly. At the same partmentalization.[6] time, the complex surface chemistry and As biochemical engineers we are inter- structure of proteins allow these molecules ested in understanding the physical deter- *Correspondence: Prof. P. Arosio to perform specific tasks by generating minants of biomolecular assembly in living ETH Zurich highly specific intermolecular interactions organisms and their connection with func- Department of Chemistry and Applied Biosciences with other proteins and biomolecules. tional and aberrant behaviors. This activity Institute for Chemical and Bioengineering, Vladimir Prelog Weg 1, CH-8093 Zurich This subtle balance of intermolecular requires addressing a series of theoretical E-mail: [email protected] forces is highly sensitive to environmental and analytical challenges, since biological News from New Chemistry Professors iN switzerlaNd CHIMIA 2018, 72, No. 5 305 systems consist of heterogeneous mixtures the generation of the soluble intermediates. of nuclei before fibril growth can occur. that often exhibit transient interactions, In analogy to traditional fields of chemical Chemical kinetic analysis has revealed that low concentration of metastable species engineering, such as polymer reaction en- each individual microscopic reaction of and complex aggregation networks. In gineering, colloidal dispersions and com- growth and secondary nucleation is actu- the following paragraphs we show how bustion processes, also in this context con- ally present from the very beginning of the concepts of chemical engineering lead cepts of reaction engineering and chemical aggregation process, and that the lag-phase to theoretical and experimental advances kinetics allow to unravel complex kinetic represents the time required for fibrils to which allow to achieve quantitative infor- schemes from a limited number of avail- amplify and reach a critical concentration mation about protein self-assembly pro- able macroscopic data (Fig. 1).[9] which is detectable by experimental ana- cesses at the molecular level. We highlight In addition to the identification of the lytical methods.[12] This concept has been the direct implications of these findings in critical microscopic steps that are most re- exploited to develop a highly sensitive as- different areas of biological and biomedi- sponsible for the generation of toxic spe- say for the quantification of low concentra- cal sciences, including the diagnosis and cies,[10] this information is fundamental to tions of amyloid fibrils.[13] therapy of Alzheimer’s disease and cell explain several aspects of the aggregation Measurements of aggregation rates can compartmentalization. We finally discuss process.[11] For instance, the application also be used to indirectly quantify specific how the developed approaches and the les- of chemical kinetic analysis has eluci- interactions between molecules by analyz- sons learned from nature allow to design dated the nature and the molecular origin ing changes in the aggregation profiles in functional materials as well as to monitor of the macroscopic lag-phase that is typi- the absence and presence of the binding and engineer the stability of proteins in cally observed in the formation of amy- partner.[14] This approach is attractive in biotechnology. loids from solutions of soluble peptides applications characterized by the presence and proteins.[12] This lag-phase could be of complex mixtures as well as transient erroneously interpreted as a waiting time and metastable interactions. One example 2. Reaction Engineering in required to reach a critical concentration is the analysis of the mechanisms of inhi- Amyloids and Alzheimer’s Disease The formation of amyloid plaques of the peptide Abeta42 (Aβ42) or analogous peptides, which originate from the frag- a mentation of the amyloid precursor protein (APP), is a hallmark of Alzheimer’s dis- ease (AD), a devastating form of dementia that affects more than one hundred thou- sand people in Switzerland and millions of people worldwide.[7] An increasing amount of evidence indicates that the formation of amyloid fibrils is a critical upstream pro- DNNAJB6 b proSP-C Brichos cess in the series of events leading to the loss of neuronal function.[8] Today no ef- Hsp70 fective pharmaceutical treatment exists for this disorder, a fact that reflects our cur- rent lack of understanding of the molecu- (ii) lar mechanisms underpinning the disease. ooligligoommeerr + ele ongation Despite the direct proof of the causality monomer secondary between the formation of aggregates and primary fibfibrilril nucleation nucleation (iii) the disease is still lacking, the aggregation (i) process represents an attractive target for therapeutic intervention. In particular, it is emerging