Characterization of Interaction Between ABBA and NEDD9

Ihmisen aivokuoren merkittäviä ominaisuuksia ovat sen suuri koko ja poimuttuneisuus. Nämä piirteet ovat laajalti alkiovaiheen kehityksen aikaisten hermokantasolujen korkean määrän sekä monimuotoisuuden ansiota. Radiaaligliasolut ovat erityisen suuressa roolissa alkion neurogeneesin aikana, sillä ne tuottavat useita eri solutyyppejä sekä toimivat mekaanisena tukena vastasyntyneille hermosoluille, jotka vaeltavat aivokuoren kehityksen aikana kohti lopullista sijaintiaan. Näille soluille tyypillisiä ominaispiirteitä ovat niiden jakautumiseen liittyvät ainutlaatuiset prosessit, kuten sooman migraatio solusyklin eri vaiheiden aikana sekä jakautumistason tarkka säätely. Vaikka näiden solujen jakautumisprosesseja on tutkittu melko laajalti, niiden taustalla vaikuttavat molekyylitason mekanismit ovat vielä suurelta osin tuntemattomia. ABBA ja NEDD9 ovat proteiineja, joiden on todettu esiintyvän radiaaligliasoluissa alkionkehityksen aikana. ABBA toimii säätelijänä solukalvon dynaamisissa muutoksissa sekä aktiinin polymerisaatiossa radiaaligliasoluissa, kun taas NEDD9-ekspressiotasoilla on osoitettu olevan suora vaikutus solujen kykyyn edetä mitoosista sytokineesiin. ABBA- ja NEDD9-proteiinien välillä on aiemmin todettu vuorovaikutus Y2H (yeast two-hybrid) -seulonnassa. Tämän pro gradu -tutkielman tavoitteena oli vahvistaa ABBA- ja NEDD9-proteiinien välinen vuorovaikutus biokemiallisesti. Ensin niiden vuorovaikutusta arvioitiin ko-immunopresipitaatiolla, joissa käytettiin C6-glioomasolujen endogeenisia proteiineja. Toinen lähestymistapa oli testata, onko näiden proteiinien välillä kyseessä välitön interaktio NEDD9:n N-terminaalisen SH3-domeenin sekä ABBA:n proliinirikkaan C-terminaalisen osan välillä. Tämä hypoteesi testattiin käyttämällä puhdistettuja proteiineja sekä domeeneja biokemiallisissa sitoutumisanalyyseissä. Vaikka ko-immunopresipitaatio-analyyseissä ilmeni selkeä vuorovaikutus, kykenin osoittamaan, ettei NEDD9 SH3-domeenin ja ABBA:n välillä ole suoraa sitoutumista. Tämä viittaa siihen, että vuorovaikutus voi vaatia muita domeeneja tai olla epäsuora. Nämä tulokset tarjoavat uutta tietoa, joka auttaa karakterisoimaan ABBA:n ja NEDD9:n biologista merkityksistä aivokuoren kehityksessä sekä muissa konteksteissa.The human cerebral cortex is characteristically large and folded, which can be majorly attributed to the high number and variety of neural progenitors during embryonic development. Radial glial cells are essential neural progenitors during neurogenesis. In addition to giving rise to new cell types, they also provide scaffold for migrating newborn neurons. Radial glia are known to portray peculiar characteristics in their cell division process, including unique migratory behavior as well as specifically regulated cleavage furrow orientation. While these processes of radial glial division have been studied extensively, the underlying molecular mechanisms are still largely unknown. ABBA (actin-bundling protein with BAIAP2 homology) and NEDD9 (neural precursor cell expressed, developmentally downregulated 9) are proteins, which are both known to be expressed in certain radial glia progenitors during embryonic development, while they are mainly absent in neurons. ABBA has a defined role of regulating plasma membrane deformation and actin polymerization in radial glia, while NEDD9 expression levels are a known factor in the correct progression from mitosis to cytokinesis. An interaction between ABBA and NEDD9 has previously been identified in a yeast two-hybrid screen done for the embryonic mouse brain. The aim of this thesis was to validate the interaction between ABBA and NEDD9 biochemically. First, their interaction was evaluated by doing co-immunoprecipitation assays on the endogenous proteins from C6 cells. The second approach was to test, whether their interaction is directly mediated by the N-terminal SH3-domain of NEDD9 and the proline-rich C-terminal portion of ABBA. This was done by doing biochemical binding assays using purified proteins and domains of interest. While co-immunoprecipitation of the two proteins gave results indicating an interaction, I could show that there is no direct binding between NEDD9 SH3-domain and ABBA, suggesting that the interaction might require other domains or be indirect. Together, these results provide valuable information that will help characterize what roles of ABBA and NEDD9 play in cortical development and beyond

Global analysis of aging-related protein structural changes uncovers enzyme-polymerization-based control of longevity

Aging is associated with progressive phenotypic changes. Virtually all cellular phenotypes are produced by proteins, and their structural alterations can lead to age-related diseases. However, we still lack comprehensive knowledge of proteins undergoing structural-functional changes during cellular aging and their contributions to age-related phenotypes. Here, we conducted proteome-wide analysis of early age-related protein structural changes in budding yeast using limited proteolysis-mass spectrometry (LiP-MS). The results, compiled in online ProtAge catalog, unraveled age-related functional changes in regulators of translation, protein folding, and amino acid metabolism. Mechanistically, we found that folded glutamate synthase Glt1 polymerizes into supramolecular self-assemblies during aging, causing breakdown of cellular amino acid homeostasis. Inhibiting Glt1 polymerization by mutating the polymerization interface restored amino acid levels in aged cells, attenuated mitochondrial dysfunction, and led to lifespan extension. Altogether, this comprehensive map of protein structural changes enables identifying mechanisms of age-related phenotypes and offers opportunities for their reversal.Peer reviewe

Global analysis of aging-related protein structural changes uncovers enzyme-polymerization-based control of longevity

Aging is associated with progressive phenotypic changes. Virtually all cellular phenotypes are produced by proteins, and their structural alterations can lead to age-related diseases. However, we still lack comprehensive knowledge of proteins undergoing structural-functional changes during cellular aging and their contributions to age-related phenotypes. Here, we conducted proteome-wide analysis of early age-related protein structural changes in budding yeast using limited proteolysis-mass spectrometry (LiP-MS). The results, compiled in online ProtAge catalog, unraveled age-related functional changes in regulators of translation, protein folding, and amino acid metabolism. Mechanistically, we found that folded glutamate synthase Glt1 polymerizes into supramolecular self-assemblies during aging, causing breakdown of cellular amino acid homeostasis. Inhibiting Glt1 polymerization by mutating the polymerization interface restored amino acid levels in aged cells, attenuated mitochondrial dysfunction, and led to lifespan extension. Altogether, this comprehensive map of protein structural changes enables identifying mechanisms of age-related phenotypes and offers opportunities for their reversal.ISSN:1097-2765ISSN:1097-416