Genetically encoded photocross-linkers determine the biological binding site of exendin-4 peptide in the N-terminal domain of the intact human glucagon-like peptide-1 receptor (GLP-1R)

The glucagon-like peptide-1 receptor (GLP-1R) is a key therapeutic target in the management of type II diabetes mellitus, with actions including regulation of insulin biosynthesis and secretion, promotion of satiety, and preservation of β-cell mass. Like most class B G protein-coupled receptors (GPCRs), there is limited knowledge linking biological activity of the GLP-1R with the molecular structure of an intact, full-length, and functional receptor·ligand complex. In this study, we have utilized genetic code expansion to site-specifically incorporate the photoactive amino acid p-azido-l-phenylalanine (azF) into N-terminal residues of a full-length functional human GLP-1R in mammalian cells. UV-mediated photolysis of azF was then carried out to induce targeted photocross-linking to determine the proximity of the azido group in the mutant receptor with the peptide exendin-4. Cross-linking data were compared directly with the crystal structure of the isolated N-terminal extracellular domain of the GLP-1R in complex with exendin(9–39), revealing both similarities as well as distinct differences in the mode of interaction. Generation of a molecular model to accommodate the photocross-linking constraints highlights the potential influence of environmental conditions on the conformation of the receptor·peptide complex, including folding dynamics of the peptide and formation of dimeric and higher order oligomeric receptor multimers. These data demonstrate that crystal structures of isolated receptor regions may not give a complete reflection of peptide/receptor interactions and should be combined with additional experimental constraints to reveal peptide/receptor interactions occurring in the dynamic, native, and full-length receptor state

Umhverfi - listaverk - áhorfandi. Skúlptúrlistaverk Richard Serra og Yoko Ono í Viðey og tengsl þeirra við umhverfi og áhorfanda.

Í ritgerðinni er fjallað um listaverk tveggja erlendra listamanna sem staðsett eru í Viðey. Annars vegar er þar að finna verk Richard Serra, Áfangar (1990) og hins vegar verk Yoko Ono, Friðarsúlan (2007). Listamennirnir eru mjög svo ólíkir en eiga það þó sameiginlegt að útlit, efni og inntak verka þeirra má rekja til breytinga sem varð á listforminu um miðja síðustu öld. Staðsetning verkanna gefur möguleika á ítarlegri greiningu sambands þeirra við umhverfi sitt og samanburðar þeirra á milli. Þá eru verkin í opinberu rými og því kostur á að rannsaka þau í tengslum við áhorfandann. Í ritgerðinni eru verkin greind og borin saman í ljósi sambands þessara þriggja þátta, umhverfis, listaverks og áhorfanda. Þær hugmyndir sem liggja verkunum til grundvallar og fjalla um samband hinna þriggja þátta með einum eða öðrum hætti eru reifaðar. Sérstaklega er litið til skúlptúrlistar og hugmyndarinnar um staðbundin(e. site-specific) verk og það sem kalla mætti þátttökulist (e. Relational art). Einnig eru listferlar listamannanna raktir og settir í samhengi stefna og strauma. Þá er listaverkunum, tilurð þeirra og framsetningu gerð ítarleg skil og fjallað um staðsetningu þeirra í Viðey. Með þetta að leiðarljósi er ritgerðinni ætlað að svara því hvernig staðsetningin á þátt í merkingarsköpun verkanna með tiliti til útlits, efnis eða inntaks og hvort og þá hvernig verkin eru staðbundin. Til þess er stuðst við hugmyndir Kenneth Framptons um Critical Regionalism, en staðfræði umhverfisins er einmitt ein forsenda kenningar Framptons. Þá eru hugleiðingar um hvernig þátttaka áhorfandans bætir við merkingu verksins til viðbótar við merkinguna sem ræðst af sambandi þess við staðsetninguna. Að lokum er því svarað hvernig sambandi þáttanna þriggja, umhverfis, listaverks og áhorfanda, er háttað í verkunum tveimur. Eðli sambandsins má hafa að leiðarljósi þegar meta skal listrænt gildi umhverfislistaverka. Með því að svara þessum spurningum þá verða til aðrar sem snúa að ytri ramma þáttanna þriggja, þ.e. almennari þáttum og tilhneigingu í nútíma skúlptúrlist, hlutverki Reykjavíkurborgar í mótun og endanlegri útkomu verkanna og á hvern hátt verkunum er ætlað hlutverk og notagildi í samhengi opinbers rýmis borgarinnar

Analys av upplägg och investeringsprocesser inom universitetsanknutna riskkapitalbolag

With the increasing importance of the third mission of universities, namely the commercialisation of science and research, universities have established various mechanisms such as technology transfer offices, business incubators and more. Since many investors tend not to invest in university spin-offs and start-ups due to the risk associated with their early stage, universities have established their own investment units, referred to as university-affiliated venture capital, to provide funding for university spin-offs and start-ups. As there is little academic research on the structure and functioning of these investment units, this paper contributes to this research by analysing the structure and investment process of university-affiliated venture capitals. For this purpose, 10 investment units in Denmark, Finland, the Netherlands, Norway, and Sweden were analysed. Among the key findings of this work is the recognition that UVCs essentially look for the abilities ofthe founders and the possibility of developing them into an effective team when selecting ventures. Inaddition, the financing instruments offered, and the ticket sizes of the investment units could be identified. While many UVCs use both equity investment and convertible loans, there is a slight bias towards convertible loans as the potential target companies are at such an early stage that valuations are very difficult to conduct and the UVCs have limited resources. Ticket sizes generally vary between 25,000 and 100,000 thousand euros.Med den ökande betydelsen av universitetens tredje uppgift, nämligen kommersialisering av vetenskap och forskning, har universiteten inrättat olika mekanismer som tekniköverföringskontor och företagsinkubatorer. Eftersom många investerare tenderar att inte investera i universitetsavknoppningar och nystartade företag på grund av den risk som är förknippad med deras tidiga skede, har universiteten inrättat sina egna investeringsenheter, så kallat universitetsanknutet riskkapital (UVC), för att tillhandahålla finansiering för universitetsavknoppningar och nystartade företag. Eftersom det inte finns så mycket akademisk forskning om strukturen och funktionen hos dessa investeringsenheter, bidrar denna uppsats till denna forskning genom att analysera strukturen och investeringsprocessen för universitetsanknutna riskkapitalbolag. För detta ändamål analyserades tio investeringsenheter; i Danmark, Finland, Nederländerna, Norge och Sverige. Bland de viktigaste resultaten av detta arbete finns insikten att UVC:er i huvudsak letar efter grundarnas förmågor och möjligheten att utveckla dem till ett effektivt team när de väljer ut företag. Dessutom kunde de finansieringsinstrument som erbjuds och investeringsstorlekarna identifieras. Även om många UVC:er använder både kapitalinvesteringar och konvertibla lån, finns det en viss övervikt för konvertibla lån eftersom de potentiella målföretagen befinner sig i ett så tidigt skede att värderingar är mycket svåra att genomföra och UVC:er har begränsade resurser. Investeringsstorlekarna varierar i allmänhet mellan 25 000 och 100 000 tusen euro

Analys av upplägg och investeringsprocesser inom universitetsanknutna riskkapitalbolag

With the increasing importance of the third mission of universities, namely the commercialisation of science and research, universities have established various mechanisms such as technology transfer offices, business incubators and more. Since many investors tend not to invest in university spin-offs and start-ups due to the risk associated with their early stage, universities have established their own investment units, referred to as university-affiliated venture capital, to provide funding for university spin-offs and start-ups. As there is little academic research on the structure and functioning of these investment units, this paper contributes to this research by analysing the structure and investment process of university-affiliated venture capitals. For this purpose, 10 investment units in Denmark, Finland, the Netherlands, Norway, and Sweden were analysed. Among the key findings of this work is the recognition that UVCs essentially look for the abilities ofthe founders and the possibility of developing them into an effective team when selecting ventures. Inaddition, the financing instruments offered, and the ticket sizes of the investment units could be identified. While many UVCs use both equity investment and convertible loans, there is a slight bias towards convertible loans as the potential target companies are at such an early stage that valuations are very difficult to conduct and the UVCs have limited resources. Ticket sizes generally vary between 25,000 and 100,000 thousand euros.Med den ökande betydelsen av universitetens tredje uppgift, nämligen kommersialisering av vetenskap och forskning, har universiteten inrättat olika mekanismer som tekniköverföringskontor och företagsinkubatorer. Eftersom många investerare tenderar att inte investera i universitetsavknoppningar och nystartade företag på grund av den risk som är förknippad med deras tidiga skede, har universiteten inrättat sina egna investeringsenheter, så kallat universitetsanknutet riskkapital (UVC), för att tillhandahålla finansiering för universitetsavknoppningar och nystartade företag. Eftersom det inte finns så mycket akademisk forskning om strukturen och funktionen hos dessa investeringsenheter, bidrar denna uppsats till denna forskning genom att analysera strukturen och investeringsprocessen för universitetsanknutna riskkapitalbolag. För detta ändamål analyserades tio investeringsenheter; i Danmark, Finland, Nederländerna, Norge och Sverige. Bland de viktigaste resultaten av detta arbete finns insikten att UVC:er i huvudsak letar efter grundarnas förmågor och möjligheten att utveckla dem till ett effektivt team när de väljer ut företag. Dessutom kunde de finansieringsinstrument som erbjuds och investeringsstorlekarna identifieras. Även om många UVC:er använder både kapitalinvesteringar och konvertibla lån, finns det en viss övervikt för konvertibla lån eftersom de potentiella målföretagen befinner sig i ett så tidigt skede att värderingar är mycket svåra att genomföra och UVC:er har begränsade resurser. Investeringsstorlekarna varierar i allmänhet mellan 25 000 och 100 000 tusen euro

Mapping the Binding Site for Escitalopram and Paroxetine in the Human Serotonin Transporter Using Genetically Encoded Photo-Cross-Linkers

In spite of the important role of the human serotonin transporter (hSERT) in depression treatment, the molecular details of how antidepressant drugs bind are still not completely understood, in particular those related to potential high- and low-affinity binding sites in hSERT. Here, we utilize amber codon suppression in hSERT to encode the photo-cross-linking unnatural amino acid <i>p</i>-azido-l-phenylalanine into the suggested high- and low-affinity binding sites. We then employ UV-induced cross-linking with azF to map the binding site of escitalopram and paroxetine, two prototypical selective serotonin reuptake inhibitors (SSRIs). We find that the two antidepressant drugs exclusively cross-link to azF incorporated at the high-affinity binding site of hSERT, while cross-linking is not observed at the low-affinity binding site. Combined with previous homology models and recent structural data on hSERT, our results provide important information to understand the molecular details of these clinical relevant binding sites

Importance of the Extracellular Loop 4 in the Human Serotonin Transporter for Inhibitor Binding and Substrate Translocation

The serotonin transporter (SERT) terminates serotonergic neurotransmission by performing reuptake of released serotonin, and SERT is the primary target for antidepressants. SERT mediates the reuptake of serotonin through an alternating access mechanism, implying that a central substrate site is connected to both sides of the membrane by permeation pathways, of which only one is accessible at a time. The coordinated conformational changes in SERT associated with substrate translocation are not fully understood. Here, we have identified a Leu to Glu mutation at position 406 (L406E) in the extracellular loop 4 (EL4) of human SERT, which induced a remarkable gain-of-potency (up to >40-fold) for a range of SERT inhibitors. The effects were highly specific for L406E relative to six other mutations in the same position, including the closely related L406D mutation, showing that the effects induced by L406E are not simply charge-related effects. Leu(406) is located >10 Å from the central inhibitor binding site indicating that the mutation affects inhibitor binding in an indirect manner. We found that L406E decreased accessibility to a residue in the cytoplasmic pathway. The shift in equilibrium to favor a more outward-facing conformation of SERT can explain the reduced turnover rate and increased association rate of inhibitor binding we found for L406E. Together, our findings show that EL4 allosterically can modulate inhibitor binding within the central binding site, and substantiates that EL4 has an important role in controlling the conformational equilibrium of human SERT

Genetically encoded photocrosslinkers locate the high-affinity binding site of antidepressant drugs in the human serotonin transporter

Despite the well-established role of the human serotonin transporter (hSERT) in the treatment of depression, the molecular details of antidepressant drug binding are still not fully understood. Here we utilize amber codon suppression in a membrane-bound transporter protein to encode photocrosslinking unnatural amino acids (UAAs) into 75 different positions in hSERT. UAAs are incorporated with high specificity, and functionally active transporters have similar transport properties and pharmacological profiles compared with wild-type transporters. We employ ultraviolet-induced crosslinking with p-azido-L-phenylalanine (azF) at selected positions in hSERT to map the binding site of imipramine, a prototypical tricyclic antidepressant, and vortioxetine, a novel multimodal antidepressant. We find that the two antidepressants crosslink with azF incorporated at different positions within the central substrate-binding site of hSERT, while no crosslinking is observed at the vestibular-binding site. Taken together, our data provide direct evidence for defining the high-affinity antidepressant binding site in hSERT