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Amyloid Beta – Biotherapy Target and Biotechnological Tool
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. (Stefan Ståhl)ORCID iD: 0000-0003-0701-3800
2020 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Alzheimer’s disease is a progressive neurodegenerative disease characterized primarily by memory impairment and cognitive decline. As of 2020, an estimated 50 million people suffer from Alzheimer’s disease or related dementia and no disease-modifying treatment options are yet approved for clinical practice. A major pathological feature of Alzheimer’s disease is the presence of cerebral senile plaques with aggregated amyloid beta (Aβ) peptides as the main constituent. In this thesis, Aβ is used in five separate studies either as a target for the development of potential biotherapeutical interventions or as a tool in biotechnological research.

In the first study, a high-throughput screening method was developed that enables functiondriven selection of protein-based aggregation inhibitors from combinatorial libraries. The method employs a reporter protein consisting of Aβ42 fused to the N-terminus of green fluorescent protein (GFP). The reporter protein misfolds due to the aggregating nature of Aβ42. Following protein expression in Escherichia coli a low whole-cell GFP fluorescence signal was detected using flow cytometry. However, when co-expressed with an affibodybased aggregation inhibitor, the reporter protein was rescued from aggregation and an increased whole-cell GFP fluorescence signal was detected in flow cytometry. By combining the screening method with flow cytometric cell sorting, the aggregation-inhibiting affibody molecule could successfully be enriched from a large background of non-inhibiting affibody molecules. The results thus demonstrated that the developed method enables highthroughput screening and sorting of combinatorial protein libraries based on the Aβ aggregation inhibiting ability.

The second study explored a strategy to increase the uptake of a biotherapeutical candidate protein into the central nervous system (CNS) via receptor-mediated transcytosis across the blood-brain barrier (BBB). The affibody-based candidate ZSYM73 binds monomeric Aβ and inhibits Aβ aggregation. Here, ZSYM73 was fused to the C-terminus of a single-chain variable fragment (scFv8D3) binding the transferrin receptor (TfR); a receptor expressed on the BBB. An engineered albumin-binding domain (ABD) was fused to ZSYM73 to extend the circulatory half-life of the fusion protein. In a mouse study, the tri-specific fusion protein scFv8D3-ZSYM73-ABD exhibited increased cerebrospinal fluid (CSF) bioavailability compared to the control protein ZSYM73-ABD, indicating an active transport mechanism into the CNS.

In the third study, a novel method for combinatorial protease engineering was developed and applied to generate highly proteolytic active variants of the coxsackievirus 3C protease. The method is based on the findings form the first study and employs a reporter protein consisting of Aβ42 fused to the N-terminus of GFP via a peptide linker containing a protease substrate sequence. The reporter protein misfolds upon expression in E. coli, which resulted in a low whole-cell GFP fluorescence signal detected in flow cytometry. Co-expression of a protease with activity on the substrate sequence led to proteolytic separation of the aggregation-prone Aβ42 peptide from GFP and restored whole-cell fluorescence. This method was used in combination with flow cytometric cell sorting to isolate highly proteolytic active variants from a randomly mutated 3C protease library. The aim of the fourth study was to evaluate the potential of the newly developed method from the third study to engineer the substrate specificity of proteases. A semi-rational tobacco etch virus (TEV) protease library was screened for variants with proteolytic activity on a novel target substrate. The target substrate differed substantially from the wild-type TEV consensus substrate and exhibited high sequence similarity to the aggregation-inducing hydrophobic core region of Aβ. After three rounds of flow cytometric cell sorting, a set of TEV protease variants was enriched that exhibited proteolytic activity on the novel substrate.

In the fifth study, a methodology employing flow cytometric sorting of multiprotein aggregates was developed to investigate the protein interactome related to Aβ plaques. It was demonstrated that in human serum or human CSF, Aβ aggregates bound to a fluorescent probe can be detected and isolated using flow cytometry. Quantitative mass spectrometry analysis was performed on Aβ aggregates isolated from human CSF. The abundances and functional features of proteins found in the isolated aggregates were investigated, and a hypothetical model of the layered architecture of Aβ aggregates was proposed. 

In conclusion, this thesis describes the development of new concepts and methods that will hopefully contribute to increasing the understanding and improving the therapy of Alzheimer’s disease and other diseases associated with protein aggregation. 

Abstract [sv]

Allt liv som vi känner till är till högsta grad beroende av proteiner. Proteiner utför nästan alla biologiska processer inom alla livsdomäner. Proteiner ger struktur till våra celler, transporterar syre i vårt blod, förser oss med energi genom att bryta ner socker och dessutom skyddar de oss från smittsamma sjukdomar, för att bara nämna några exempel. Proteiner består av kedjor av sammanlänkade aminosyror. De flesta proteiner består av ett unikt antal och en specifik ordning av 20 olika aminosyror. Aminosyrakedjorna veckas oftast till tredimensionella proteinstrukturer som gör att de kan utföra sina biologiska funktioner. Följaktligen kan det vara skadligt när veckningsprocessen går fel och proteiner inte antar deras funktionella struktur. Felveckade proteiner associeras med en rad olika sjukdomar. Ett sådant exempel är Alzheimers sjukdom, en irreversibel, progressiv hjärnsjukdom som gradvis leder till försämrat minne och mental förmåga. Psykiska och andra neurologiska symtom är även mycket vanliga och sjukdomen leder ofta till en för tidig död. I hjärnorna hos patienter med Alzheimers sjukdom bildas små klumpar, så kallade senila plack, som består av felveckade och aggregerade proteiner. Huvudbeståndsdelen i dessa klumpar är ett protein som kallas amyloid beta (Aβ).

Protein engineering beskriver processen att generera proteiner med förbättrade eller nya funktioner anpassade efter önskade egenskaper. Idag används såna proteiner inom olika områden, till exempel jordbruk- och livsmedelsindustri, diagnostik- och läkemedelsindustri och biobränsleproduktion. Affinitetsproteiner och proteaser är två typer av proteiner som har varit centrala för studierna i den här avhandlingen. Deras förmåga att specifikt binda till respektive klyva andra målproteiner har lett till en utbredd användning som bland annat forskningsreagens men även som molekyler för terapeutiska ändamål.

I studierna som ligger till grund för den här avhandlingen har amyloid beta dels använts som ett målprotein för att utveckla potentiella terapeutiska proteiner, men även som ett molekylärt verktyg för utveckling av nya metoder för protein engineering, och slutligen för att studera sammansättningen av patologiska proteinaggregat från Alzheimerpatienter.

Den första studien i denna avhandling beskriver utvecklingen av en ny metod för protein engineering för att effektivisera utvecklingen av aggregeringshämmare. Hämningen av Aβaggregering och följaktligen bildning av senila plack är en potentiell mekanism för att behandla Alzheimers sjukdom. I den andra studien konstruerades ett bioterapeutiskt kandidatprotein, som binder till Aβ, för att förbättra upptaget till centrala nervsystemet. Ökat läkemedelsupptag i centrala nervsystemet, platsen där amyloid beta aggregerar, har en stor sannolikhet att öka läkemedlets terapeutiska effekt. I den tredje studien utvecklades en ny metod för att generera proteinklyvande proteaser med förbättrade eller nya funktioner. Denna metod är baserad på aggregering av Aβ-peptiden och genererade ett antal nya proteasvarianter med förbättrad förmåga att klyva ett målprotein. I den fjärde studien användes samma metod för att generera proteasvarianter som klyver ett nytt målprotein som är väldigt likt Aβ. De nya varianterna har potential att vidareutvecklas till proteasbaserade läkemedelskandidater i framtiden. Den femte studien beskriver utvecklingen av en metod för att undersöka sammansättningen av tidigare nämnda senila plack. Den nya kunskapen om sammansättning av plack kan bidra till att förstå sjukdomens patologi, och utveckling av potentiella nya terapier.

Sammanfattningsvis så kommer resultaten som presenteras i denna avhandling förhoppningsvis att bidra till ökad förståelse och förbättrad behandling av Alzheimers sjukdom och andra sjukdomar som associeras med proteinaggregering.

Place, publisher, year, edition, pages
KTH Royal Institute of Technology, 2020.
Keywords [en]
Alzheimer’s disease, amyloid beta, blood-brain barrier, green-fluorescent protein, intracellular assays, proteases, protease substrate specificity, protein aggregation, protein engineering.
National Category
Natural Sciences
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-272826ISBN: 978-91-7873-507-5 (print)OAI: oai:DiVA.org:kth-272826DiVA, id: diva2:1427220
Public defence
2020-05-29, https://kth-se.zoom.us/webinar/register/WN_QqN3EAF1RvagtaZIodM8Tw, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

QC 2020-05-06

Available from: 2020-05-06 Created: 2020-04-29 Last updated: 2020-05-06Bibliographically approved
List of papers
1. Flow-cytometric screening of aggregation-inhibitors using a fluorescence-assisted intracellular method
Open this publication in new window or tab >>Flow-cytometric screening of aggregation-inhibitors using a fluorescence-assisted intracellular method
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2017 (English)In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 12, no 1, article id 1600364Article in journal (Refereed) Published
Abstract [en]

Aggregation of misfolded peptides and proteins is a key event in several neurodegenerative diseases. Suggested treatments of such disorders aim to inhibit the initial aggregation process. Here, we have developed an intracellular, function-based screening method, intended for isolation of aggregation-inhibitors from combinatorial protein libraries by flow-cytometric cell sorting. The method is based on fusion of aggregation-prone peptides to a fluorescent protein, functioning as a solubility reporter. Co-expression of a protein-based aggregation-inhibitor should prevent aggregation and thus increase the whole-cell fluorescence. We evaluated the method using the aggregation-prone Alzheimer's-related amyloid-β (Aβ) peptide in fusion to green-fluorescent protein (GFP), and an Aβ aggregation-inhibiting Affibody molecule. To adapt the method for library applications, the inhibitor was linked to an mCherry reporter for normalization of protein expression levels. We found that aggregation propensity correlates with fluorescence intensity, as co-expression of the Affibody-inhibitor increased the whole-cell fluorescence relative to a non-inhibitor. Employing improved cultivation parameters, we furthermore demonstrated efficient rescue from aggregation of an α-synuclein-derived protein using a different type of aggregation-inhibitor. Importantly, we also showed that the Aβ aggregation-inhibiting Affibody molecule could be isolated from a 1:10,000 background of non-inhibitors, with around 3,500-fold enrichment, in one cycle of fluorescence-based cell sorting. In conclusion, our new method represents a promising approach for generation of novel protein-based aggregation-inhibitors.

Place, publisher, year, edition, pages
John Wiley & Sons, 2017
Keywords
Affibody molecules, Aggregation-inhibitor, Amyloid beta, Combinatorial protein engineering, Intracellular selection system, Cells, Cytology, Diagnosis, Fluorescence, Glycoproteins, Molecules, Neurodegenerative diseases, Peptides, Aggregation inhibitors, Amyloid betas, Protein engineering, Selection systems, Proteins, alpha synuclein, amyloid beta protein, green fluorescent protein, recombinant protein, chemistry, Escherichia coli, flow cytometry, gene vector, genetics, metabolism, preclinical study, procedures, alpha-Synuclein, Amyloid beta-Peptides, Drug Evaluation, Preclinical, Genetic Vectors, Green Fluorescent Proteins, Recombinant Proteins
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-202248 (URN)10.1002/biot.201600364 (DOI)000395638400009 ()27860405 (PubMedID)2-s2.0-85006293410 (Scopus ID)
Note

Funding text: We thank Affibody AB for providing the genetic construct of ZHER2-mCherry. The Wallenberg Center for Protein Research and the Swedish Brain Foundation (grant FO2015-0174) are acknowledged for funding. Conceived and designed the experiments: HL LS JL SS. Performed the experiments: HL LS SM. Contributed reagents/materials/analysis tools and analyzed the data: HL LS SM MU JL SS. Wrote the paper: HL LS JL SS. The authors declare no conflicts of interest. QC 20170313

Available from: 2017-03-13 Created: 2017-03-13 Last updated: 2020-04-29Bibliographically approved
2. An Affibody Molecule Is Actively Transported into the Cerebrospinal Fluid via Binding to the Transferrin Receptor
Open this publication in new window or tab >>An Affibody Molecule Is Actively Transported into the Cerebrospinal Fluid via Binding to the Transferrin Receptor
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2020 (English)In: International Journal of Molecular Sciences, ISSN 1422-0067, E-ISSN 1422-0067Article in journal (Refereed) Published
Abstract [en]

Theuseofbiotherapeuticsforthetreatmentofdiseasesofthecentralnervoussystem(CNS) is typically impeded by insufficient transport across the blood–brain barrier. Here, we investigate a strategy to potentially increase the uptake into the CNS of an affibody molecule (ZSYM73) via binding to the transferrin receptor (TfR). ZSYM73 binds monomeric amyloid beta, a peptide involved in Alzheimer’s disease pathogenesis, with subnanomolar affinity. We generated a tri-specific fusion protein by genetically linking a single-chain variable fragment of the TfR-binding antibody 8D3 and an albumin-binding domain to the affibody molecule ZSYM73. Simultaneous tri-specific target engagementwasconfirmedinabiosensorexperimentandtheaffinityformurineTfRwasdetermined to 5 nM. Blockable binding to TfR on endothelial cells was demonstrated using flow cytometry and in a preclinical study we observed increased uptake of the tri-specific fusion protein into the cerebrospinal fluid 24 h after injection.

Keywords
neurodegenerativedisorders;affibodymolecules;blood–brainbarrier;receptor-mediated transcytosis; transferrin receptor
National Category
Pharmaceutical Biotechnology
Identifiers
urn:nbn:se:kth:diva-272824 (URN)10.3390/ijms21082999 (DOI)2-s2.0-85083854339 (Scopus ID)
Note

QC 20200505

Available from: 2020-04-29 Created: 2020-04-29 Last updated: 2020-05-05Bibliographically approved
3. Directed evolution of the 3C protease from coxsackievirus using a novel fluorescence-assisted intracellular method
Open this publication in new window or tab >>Directed evolution of the 3C protease from coxsackievirus using a novel fluorescence-assisted intracellular method
2019 (English)In: Biological chemistry (Print), ISSN 1431-6730, E-ISSN 1437-4315, Vol. 400, no 3, p. 405-415Article in journal (Refereed) Published
Abstract [en]

Proteases are crucial for regulating biological processes in organisms through hydrolysis of peptide bonds. Recombinant proteases have moreover become important tools in biotechnological, and biomedical research and as therapeutics. We have developed a label-free high-throughput method for quantitative assessment of proteolytic activity in Escherichia coli. The screening method is based on co-expression of a protease of interest and a reporter complex. This reporter consists of an aggregation-prone peptide fused to a fluorescent protein via a linker that contains the corresponding substrate sequence. Cleavage of the substrate rescues the fluorescent protein from aggregation, resulting in increased fluorescence that correlates to proteolytic activity, which can be monitored using flow cytometry. In one round of flow-cytometric cell sorting, we isolated an efficiently cleaved tobacco etch virus (TEV) substrate from a 1:100 000 background of non-cleavable sequences, with around 6000-fold enrichment. We then engineered the 3C protease from coxsackievirus B3 (CVB3 3C(pro)) towards improved proteolytic activity on the substrate LEVLFQ down arrow GP. We isolated highly proteolytic active variants from a randomly mutated CVB3 3C(pro) library with up to 4-fold increase in activity. The method enables simultaneous measurement of proteolytic activity and protease expression levels and can therefore be applied for protease substrate profiling, as well as directed evolution of proteases.

Place, publisher, year, edition, pages
WALTER DE GRUYTER GMBH, 2019
Keywords
Coxsackievirus B3, FACS, GFP-fusion, intracellular assay, protease engineering, protease substrate profiling
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-245129 (URN)10.1515/hsz-2018-0362 (DOI)000458628400014 ()30521472 (PubMedID)2-s2.0-85058219114 (Scopus ID)
Note

QC 20190313

Available from: 2019-03-13 Created: 2019-03-13 Last updated: 2020-04-29Bibliographically approved
4. Engineering the substrate specificity of TEV protease towards an Aβ-cleaving enzyme
Open this publication in new window or tab >>Engineering the substrate specificity of TEV protease towards an Aβ-cleaving enzyme
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Due to their ability to catalytically cleave proteins and peptides, proteases present unique opportunities for the use in industrial, biotechnological, and therapeutic applications. The possibility to engineer proteases with redesigned substrate specificities has the potential to expand the scope of practical applications of this enzyme class. We here apply a combinatorial protease engineering screening method that links proteolytic activity to the solubility and correct folding of a fluorescent reporter protein to redesign the substrate specificity of Tobacco Etch Virus (TEV) protease. The target substrate EKLVFQA differs at three of seven positions from the TEV consensus substrate sequence and exhibits high sequence similarity to the aggregation-inducing hydrophobic core region of the amyloid beta (Aβ) peptide. Flow cytometric sorting of a semi-rational TEV protease library led to the enrichment of a set of protease variants that recognize and cleave the novel target substrate.

Keywords
Amyloid beta; GFP-fusion; intracellular assay; protease engineering; substrate specificity; TEV protease
National Category
Pharmaceutical Biotechnology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-272823 (URN)
Note

QC 20200430

Available from: 2020-04-29 Created: 2020-04-29 Last updated: 2020-04-30Bibliographically approved
5. Dissecting the structural organization of multiprotein amyloid aggregates using a bottom-up approach
Open this publication in new window or tab >>Dissecting the structural organization of multiprotein amyloid aggregates using a bottom-up approach
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2020 (English)In: ACS Chemical Neuroscience, ISSN 1948-7193, E-ISSN 1948-7193Article in journal (Refereed) Published
Abstract [en]

Deposition of fibrillar amyloid β (Aβ) in senile plaques is a pathological signature of Alzheimer's disease. However, senile plaques also contain many other components, including a range of different proteins. Although the composition of the plaques can be analyzed in post mortem tissue, knowledge of the molecular details of these multiprotein inclusions and their assembly processes is limited, which impedes the progress in deciphering the biochemical mechanisms associated with Aβ pathology. We here describe a bottom-up approach to monitor how proteins from human cerebrospinal fluid associate with Aβ amyloid fibrils to form plaque particles. The method combines flow cytometry and mass spectrometry proteomics and allowed us to identify and quantify 128 components of the captured multiprotein aggregates. The results provide insights in the functional characteristics of the sequestered proteins and reveal distinct interactome responses for the two investigated Aβ variants, Aβ(1-40) and Aβ(1-42). Furthermore, the quantitative data is used to build models of the structural organization of the multiprotein aggregates, which suggests that Aβ is not the primary binding target for all the proteins; secondary interactions account for the majority of the assembled components. The study elucidates how different proteins are recruited into senile plaques and establishes a new model system for exploring the pathological mechanisms of Alzheimer's disease from a molecular perspective.

Keywords
amyloid, Alzheimer disease, amyloid , protein-protein interaction, flow cytometry
National Category
Pharmaceutical Biotechnology
Identifiers
urn:nbn:se:kth:diva-272825 (URN)10.1021/acschemneuro.0c00110 (DOI)
Note

QC 20200505

Available from: 2020-04-29 Created: 2020-04-29 Last updated: 2020-05-05Bibliographically approved

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