Projects / Programmes
Structural basis of calcium regulation of human alpha-actinin
| Code |
Science |
Field |
Subfield |
| 1.05.00 |
Natural sciences and mathematics |
Biochemistry and molecular biology |
|
| Code |
Science |
Field |
| B000 |
Biomedical sciences |
|
| Code |
Science |
Field |
| 1.06 |
Natural Sciences |
Biological sciences |
Actin-based cytoskeleton, stress-fibres, α-actinin, calcium regulation, structural biology
Researchers (7)
Organisations (2)
Abstract
Citoskelet je ena od osnovnih strukturnih in funckijski značilnosti vseh celic. Pri evkariontih se je razvit v ekstremno kompleksno mrežo različnih filamentov, od katerih so aktinski filamenti tisti, ki nudijo največjo fleksibilnost v smislu povezave oblika-funkcija (npr. mikrovili črevesnih epitelijskih celic in nevriti živčnih celic), celičnega premikanja (npr. lamelipodiji in filopodiji migrirajočih celic) in kontrakcije (mišične celice). To raznoliko funkcionalnost omogočajo cel spekter akcesornih proteinov od katerih so najbolj pogosti α-aktinini. Obstajajo štiri izooblike α-aktininov: dve od kalcija neodvisni izoobliki, omejeni na mišične celice (α-aktinin-2 in -3), ter o dve obliki, ki se nahajata v vseh celicah (α-aktinin-1 in -4). Slednji prečno povezujeta aktinske filamente v večje strukture – tridimenzionalne mreže, kjer so aktinski filamenti orientirani v različnih smereh, in svežji tesno povezanih vzporednih aktinskih filamentov. Svežnji aktinskih filamentov delujejo kot ogrodja, ki podpirajo ali stabilizirajo celične strukture kot so fokalni celični adhezijski stiki, izrastki in stresna vlakna. Te strukture so dinamične, kar je izrednega pomena pri odzivanju celic na njihovo spremenjene pogoje v njeni notranjosti in zunanjosti. Ključen faktor pri regulaciji tvorbe in reorganizacije aktinskih svežnjev je kalcij, ki se veže na kalmodulinu podobno domeno α-aktinina-1 in -4 ter preko tega modulira sposobnost α-aktinina, da povezuje aktinske filamente v svežnje. Reverzibilno naravo tega procesa omogočajo fluktuacije v konetraciji kalcijevih ionov v citosolu, kar je ključnega pomena za dinamično reorganizacijo aktinskega citoskeleta z direktnim vplivom na druge celične strukture. Obstoječe znanje o glavnih strukturnih in funkcijskih značilnosti α-aktinonov sicer omogoča postavitev smiselnega modela njihovega delovanja, pa podrobnosti mehanizma ostajajo neznani. Šele naši nedavno objavljeni rezultati, ki so osvetlili strukturne značilnosti regulacije α-aktinina-1 preko kalcijevih ionov, omogočajo postavitev naslednje hipoteze: vezava kalcijevih ionov na kalmodulinu-podobno domeno α-aktinina-1 sproži konformacijske spremembe, ki vključujejo sosednjo aktin-vezavno domeno, onemogocijo primerno relativno orientacijo aktin-vezavnih domen in v končni fazi negativno vplivajo na sposobnost α-aktinina-1, da povezuje aktinske filamente v snope. V luči tega je naš cilj analizirati strukturo in dinamiko α-aktinina-1 z uporabo pristopa integrativne strukturne biologije, karakterizirati obliko α-aktinina-1 z in brez vezanih kalcijevih ionov preko različnih biokemijskih in biofizikalnih metod, ter potrditi hipotetični molekularni model z uporabo in vivo pristopa. Tako bomo tudi naslovili eno od osnovnih vprašanj celične biologije: kako celice dinamično reorganizirajo aktinski citoskelet enostavno s fluktuacijami koncentracije kalcijevih ionov? Podatke o α-aktininu-1 bo možno aplicirati tudi na drugo nemišično obliko, α-aktinin-4. Nadalje, ker so mutacije v genih za α-aktinine so povezane z večimi dednimi boleznimi (kongenitalna makrotrombocitopenija, fokalna segmentna glomeruloskleroza, kardiomiopatije), bodo naši rezultati pomembno prispevali k razumevanju njihovega nastanka, razvoju terapevstkih pristopov ali strategij za preprečevanje njihovega nastanka. Nenazadnje bo interdisciplinarna narava projekta združila izredno kvalitetne raziskovalne skupine, pozitivno vplivala na nadaljnje sodelovanje, tudi zunaj meja tega projekta, pospeševala izmenjavo izkušenj ter skozi izobraževalno funkcijo Katedre za biokemijo UL FKKT prispevala k širjenju znanja.
Significance for science
The basic goal of this research proposal is to investigate and explain the molecular mechanism of actin bundling by a-actinin-1 (non-muscle form), where the key regulatory role is played by calcium ions bound to a-actinin's calmodulin-like domain. We expect that this mechanism can also be applied to the other non-muscle form (a-actinin-4), which is also subject to direct regulation by calcium ions. Compared to the regulatory mechanism of calcium-independent (muscle) isoforms of a-actinin (isoforms 2 and 3), which are regulated by PIP2 binding and for which a detailed molecular mechanism has already been described, the calcium-dependent molecular workings of non-muscle isoforms is much less known, particularly at the atomic level. Since the non-muscle isoforms of a-actinin (isoforms 1 and 4) are found in every cell the regulatory mechanism underlying their role represents one of the fundamental mechanisms which is key for formation, stability and dynamics of actin cytoskeleton. At the same time, this addresses one the the basic biological questions and substantially contribute to understanding of several important biological processes (for example, tissue formation and remodelling, immune response, and cancer cell metastasis). Furthermore, our results will help to understand the mechanism behind several inherited diseases linked to genetic variants of genes, associated with formation and dynamic reorganization of actin cytoskeleton (macrothrombocytopenia, cardiomyopathies, focal segmental glomerulosclerosis).
Significance for the country
The proposed project falls within the basic research and its results will in the first line contribute to the understanding of the complex action of a-actinin-1 at the molecular level. At the same time the results will also have a practical value since they will contribute to understanding of diseases linked to genetic variants of non-muscle a-actinins, and to the development of therapies for their treatment or even strategies to prevent their initial occurence. For example, focal segmental glomerulosclerosis is a kidney disease which in its most severe form leads to kidney failure, and its development is linked to the mutations within exon 8a of non-muscle a-actinin-4 (paralogue of a-actinin-1), coding for the region between ABD and the rod domain. Mutations within this region (implicated in Ca2+-regulation of a-actinin activity) result in increased affinity of a-actinin-4 towards actin. Our results on the regulatory aspect of a-actinin-1 would undoubtedly be extremely valuable in providing a suitable explanation of the key molecular factors involved in the disease. Even more, in-depth understanding of one of such basic cellular structures as the actin cytoskeleton would undoubtedly contribute to development of novel cell-based technologies, e.g. cell-based factories and engineered macromolecular machines.
Most important scientific results
Final report
Most important socioeconomically and culturally relevant results
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