Linköping University | Department of Physics, Chemistry and Biology Master thesis, 60 hp | Chemistry Autumn 2016 - Spring 2017 | LITH-IFM-A-EX—17/3302--SE Bri2 BRICHOS domain Eukaryotic expression and importance of strictly conserved cysteine residues Lovisa Hemmingsson Examiner, Per Hammarström Supervisors, Jan Johansson & Jenny Presto Linköpings universitet | Institution för fysik, kemi och biologi Masteruppsats, 60 hp |Kemi HT 2016 - VT 2017 | LITH-IFM-A-EX—17/3302--SE Bri2 BRICHOS domain Eukaryotic expression and importance of strictly conserved cysteine residues Lovisa Hemmingsson Examinator, Per Hammarström Handledare, Jan Johansson & Jenny Presto Avdelning, institution Datum Division, Department Date Division of Chemistry 2017-06-12 Department of Physics, Chemistry and Biology Linköping University Språk Rapporttyp ISBN Language Report category Svenska/Swedish Licentiatavhandling ISRN: LITH-IFM-A-EX--17/3302--SE Engelska/English Examensarbete _________________________________________________________________ C-uppsats D-uppsats Serietitel och serienummer ISSN ________________ Övrig rapport Title of series, numbering ______________________________ _____________ URL för elektronisk version Titel Title Bri2 BRICHOS domain Eukaryotic expression and importance of strictly conserved cysteine residues Författare Author Lovisa Hemmingsson Sammanfattning Abstract Alzheimer’s disease (AD), the most common form of dementia is associated with fibril formation of amyloid-b peptides (Ab). Ab, proteolytically derived from Ab precursor protein (AbPP), is the major component of amyloid plaques in AD brains. Familial British and Danish dementias (FBD and FDD) share pathological and clinical characteristics with AD, and the underlying mechanisms are associated with amyloid formation of mutant peptides released from the Bri2 protein. Bri2 interacts with AbPP and its BRICHOS domain has been shown to delay Ab40 and Ab42 fibril formation and toxicity in vitro and in vivo. This makes Bri2 BRICHOS a promising anti-amyloid chaperone and a potential treatment strategy for AD. Furthermore, Bri2 BRICHOS possesses a general chaperone activity as it suppresses non-fibrillar aggregation of destabilized citrate synthase (CS). Recent findings show that Bri2 BRICHOS produced in E.coli can form different molecular weight assemblies, ranging from monomers to dimers and poly-disperse oligomers. The oligomers inhibit CS aggregation, whereas the monomers and dimers are more efficient against Ab42 fibrillation and neurotoxicity, respectively. The work in this thesis shows that similar Bri2 BRICHOS quaternary structures are formed in eukaryotic cells as in E.coli. Larger BRICHOS oligomers were found in cell media, derived from proteolytically processed endogenous Bri2 in SH-SY5Y cells, as well as in human embryonic kidney (HEK293) cells transfected with a Bri2 BRICHOS construct. Recombinant human Bri2 BRICHOS mutants with one or none of the two strictly conserved cysteine residues were studied. All mutant monomers become proteolytically degraded during purification, but form stable oligomers. Single Cys to Ser mutants form stable disulfide-dependent dimers that differ in ability to prevent Ab42 fibrillation, the most stable mutant (C164S) being even more efficient than the wildtype Bri2 BRICHOS dimer. This result suggests that intra or intermolecular disulfide(s) and oligomerization affect Bri2 BRICHOS stability and activity towards Ab42 fibril formation. Nyckelord Keyword Bri2 BRICHOS, Ab, Chaperone, Amyloid fibril formation, Alzheimer disease, Eukaryotic expression, Cysteine residues Bri2 BRICHOS domain Eukaryotic expression and importance of strictly conserved cysteine residues Model of Bri2 BRICHOS. Cys164 (green) and Cys223 (blue) forming an intramolecular disulfide bond. Visualized with PyMol. Model from Knight et al, 2013 and Willander et al, 2012a. Lovisa Hemmingsson Master Thesis, 60 hp Department of Physics, Chemistry and Biology, Division of Chemistry, Linköping University Department of Neurobiology, Care Sciences and Society, Division of Neurogeriatrics, Karolinska Institutet Preface The work in this thesis was performed in Jan Johansson’s group, Department of Neurobiology, Care Sciences and Society, Division of Neurogeriatrics, Karolinska Institutet. Expi293 cells were cultured and transfected at the Department of Public Health and Caring Sciences, Division of Molecular Geriatrics, Uppsala University. Circular dichroism (CD) and luminescent conjugated oligothiophene (LCO) experiments were performed at the Department of Physics, Chemistry and Biology, Division of Chemistry, Linköping University. Abstract Alzheimer’s disease (AD), the most common form of dementia is associated with fibril formation of amyloid-b peptides (Ab). Ab, proteolytically derived from Ab precursor protein (AbPP), is the major component of amyloid plaques in AD brains. Familial British and Danish dementias (FBD and FDD) share pathological and clinical characteristics with AD, and the underlying mechanisms are associated with amyloid formation of mutant peptides released from the Bri2 protein. Bri2 interacts with AbPP and its BRICHOS domain has been shown to delay Ab40 and Ab42 fibril formation and toxicity in vitro and in vivo. This makes Bri2 BRICHOS a promising anti- amyloid chaperone and a potential treatment strategy for AD. Furthermore, Bri2 BRICHOS possesses a general chaperone activity as it suppresses non-fibrillar aggregation of destabilized citrate synthase (CS). Recent findings show that Bri2 BRICHOS produced in E.coli can form different molecular weight assemblies, ranging from monomers to dimers and poly-disperse oligomers. The oligomers inhibit CS aggregation, whereas the monomers and dimers are more efficient against Ab42 fibrillation and neurotoxicity, respectively. The work in this thesis shows that similar Bri2 BRICHOS quaternary structures are formed in eukaryotic cells as in E.coli. Larger BRICHOS oligomers were found in cell media, derived from proteolytically processed endogenous Bri2 in SH-SY5Y cells, as well as in human embryonic kidney (HEK293) cells transfected with a Bri2 BRICHOS construct. Recombinant human Bri2 BRICHOS mutants with one or none of the two strictly conserved cysteine residues were studied. All mutant monomers become proteolytically degraded during purification, but form stable oligomers. Single Cys to Ser mutants form stable disulfide- dependent dimers that differ in ability to prevent Ab42 fibrillation, the most stable mutant (C164S) being even more efficient than the wildtype Bri2 BRICHOS dimer. This result suggests that intra or intermolecular disulfide(s) and oligomerization affect Bri2 BRICHOS stability and activity towards Ab42 fibril formation. Sammanfattning Alzheimers sjukdom, den vanligaste typen av demens är associerad med fibrillbildning av amyloid-b peptiden (Ab). Ab klyvs från Ab prekursor proteinet (AbPP) och är den huvudsakliga beståndsdelen i senila plack. Familjär brittisk och dansk demens är två sällsynta amyloidsjukdomar med liknande patologiska och kliniska kännetecken som Alzheimers sjukdom. Dessa sjukdomar orsakas av fibrillering av mutanta peptider från Bri2 proteinet. Bri2 interagerar med AbPP och dess BRICHOS domän har visat sig minska Ab40 och Ab42 fibrillbildning och toxicitet både in vitro och in vivo. Bri2 BRICHOS är således ett lovande anti-amyloid chaperon och en potentiell behandlingsstrategi mot Alzheimers sjukdom. Bri2 BRICHOS kan också fungera som ett traditionellt molekylärt chaperon såtillvida att den effektivt förhindrar aggregering av termo-denaturerat citrat syntas (CS). Senaste data visar att Bri2 BRICHOS producerat i E.coli kan bilda assembler av flera olika molekylvikter, allt från monomerer till dimerer och oligomerer. Oligomeren inhiberar CS aggregering medan monomeren och dimeren är mer effektiva mot neurotoxicitet respektive Ab42 fibrillering. Den här studien visar att liknande Bri2 BRICHOS kvartärstrukturer bildas i eukaryota celler som i E.coli. Bri2 BRICHOS oligomerer detekterades i cellmedia från endogent proteolytiskt processat Bri2 i SH-SY5Y celler, samt från HEK293 celler som blivit transfekterade med ett Bri2 BRICHOS konstrukt. Rekombinant Bri2 BRICHOS med en eller ingen av dem två strikt konserverade cysteinerna studerades. Alla mutanta monomerer blir proteolytiskt nedbrytna under reningen, dock bildas det stabila oligomerer. Enkelmutanterna bildar stabila disulfidbundna dimerer som skiljer sig åt i förmåga att inhibera Ab42 fibrillering, där den mest stabila mutanten till och med är mer effektiv än Bri2 BRICHOS vildtyps-dimeren. De här resultaten indikerar att både intra och intermolekylära disulfider samt oligomerisering påverkar Bri2 BRICHOS stabilitet och aktivitet gentemot Ab42 fibrillbildning. Abbreviations Ab Amyloid-b AbPP Amyloid-b precursor protein AD Alzheimer’s disease AICD AbPP intracellular domain APOE Apolipoprotein E BBB Blood brain barrier CD Circular dichroism CNS Central nervous system CS Citrate synthase ER Endoplasmic reticulum FAD Familial AD FBD Familial British dementia FBS Fetal bovine serum FDD Familial Danish dementia FPLC Fast performance liquid chromatography HEK Human embryonic kidney hFTAA Hepta-formylthiophene acetic acid Hsp Heat shock protein ILD Interstitial lung disease IMAC Immobilized metal ion affinity chromatography ITM2B Integral membrane protein 2B LCO Luminescent conjugated oligothiophene NEM N-ethylmaleimide NFT Neurofibrillary tangle PAGE Polyacrylamide gel electrophoresis PBS Phosphate buffered saline PD Parkinson’s disease ProSP-C