Isolation, characterisation and the role of complexes formed between transferrin and insulin-like growth factor-binding protein 3

Sistem faktora rasta, po strukturi i funkciji, sličnih insulinu („insulin-like growth factor“- IGF) obuhvata elemente sa primarnom ulogom u regulaciji rasta, razvoja, diferencijacije ćelija kako u fiziološkim, tako i u patofiziološkim uslovima. Pored dva funkcionalna peptidna hormona, IGF-I i IGF-II, ovaj sistem sadrži molekule koji regulišu ispoljavanje njihove fiziološke funkcije. Regulatorni elementi IGF sistema su: vezujući proteini (IGFBP-1 do -6), receptori za IGF molekule: IGF-1R, IGF-2R, insulinski receptor (IR), hibridni receptor IGF-R/IR, kao i specifične proteaze. Nakon proteolize IGFBP, funkcija IGF-I/II se ispoljava interakcijom sa IGF specifičnim receptorima na površini ćelija. U kompleksu sa IGFBP-3 se nalazi 70-75% IGF peptida. Pored primarne uloge nosača IGF molekula, IGFBP-3 učestvuje i u brojnim interakcijama nezavisno od IGF. IGFBP-3 poseduje više strukturnih domena što ga čini sposobnim za vezivanje za druge molekule i pokretanje signalnih puteva i mehanizama nezavisnih, a nekad i suprotnih, dejstvu IGF peptida poput stimulacije rasta ćelija ili pokretanja apoptotskih procesa. Jedan od partnera koji interaguje sa IGFBP-3 je transferin (Tf), glavni transporter jona gvožđa u krvi. Iako se za postojanje kompleksa IGFBP-3/Tf znalo i ranije, rezultati ove disertacije predstavljaju prvi prikaz strukturnih i funkcionalnih osobina ovih kompleksa, kao i sagledavanje njihove moguće uloge. U ovoj disertaciji je opisan optimizovan postupak za izolovanje kompleksa iz fizioloških uzoraka (seruma i tkiva), navedene su strukturne karakteristike kompleksa IGFBP-3/Tf, faktori koji utiču na njihovo formiranje, određena je koncentracija kompleksa u serumu zdravih odraslih osoba i osoba sa poremećajem u metabolizmu gvožđa, dokazana je neophodnost jona gvožđa za formiranje kompleksa i analizirana je subćelijska raspodela kompleksa u tkivu debelog creva (zdravog i tumorskog). U radu je ispitan uticaj: anemije, visoke koncentracije gvožđa i tumora debelog creva, na koncentraciju i strukturu kompleksa. Za tumor debelog creva je karakteristična sistemska anemija, praćena akumulacijom gvožđa u samom tumorskom tkivu. Na model sistemu tumora debelog creva ispitana je važnost puta internalizacije IGFBP-3 preko kompleksa sa Tf, odnosno posredstvom transferinskog receptora (TfR). Nađeno je da je koncentracija kompleksa IGFBP-3/Tf kod zdravih ljudi 241 ± 62 μg/L i da se u ovoj formi nalazi 5-7% ukupnog IGFBP-3. Kod različitih patofizioloških stanja sa poremećajem u metabolizmu gvožđa utvrđeno je da formiranje kompleksa primarno zavisi od koncentracije gvožđa, potom koncetracije IGFBP-3, ali i proteina uključenih u metabolizam gvožđa, poput feritina. Kod IGFBP-3/Tf kompleksa analiziranih u uzorcima seruma dobijenih od pacijenata sa tumorom debelog creva primećen je izmenjen način...The insulin-like growth factor (IGF) system plays an important role in the regulation of cell growth, development and differentiation, in both physiological and pathophysiological condition. Beside two peptide hormones IGF-I and IGF-II, this system includes the following regulatory elements: six IGF-binding proteins (IGFBP-1 to -6), specific receptors for IGF (IGF-1R and IGF-2R), insulin receptor (IR), hybrid receptor (IGF-R/IR) as well as IGFBP specific proteases. When IGFBPs are proteolytically cleaved, IGF-I/II are able to exert their physiological function by interacting with specific receptors on the cell surface. Approximately 70-75% of the total IGFs, are transported in circulation in the form of ternary complexes with IGFBP-3. Besides being the principal carrier of IGF molecules, IGFBP-3 was reported to exert a number of activities which are IGF-independent. Due to its structural sub-domain organisation, this molecule is able to interact with binding partners other than IGFs, and consequently activate mechanisms and signaling cascades with independent or even opposite effects to those of IGF-I/II, such as apoptosis. One of these binding partners is transferrin (Tf), the principal iron transporter in the blood. Although the existence of IGFBP-3/Tf complexes has already been reported, the results presented in this thesis offer the first complete structural and functional characterisation of the complexes, together with the analysis of their potential role. The fully optimised method for the isolation of intact IGFBP-3/Tf complexes from serum and tissue samples has been described, some structural characteristics have been defined, the effect of iron and other factors on the formation of complexes was studied, their concentration in sera from healthy persons and patients with impaired iron metabolism disorders was measured, as well as their subcellular distribution in colon tissue (healthy and tumor tissue). The formation and concentration of IGFBP-3/Tf complexes in persons with anemia, persons with very high iron concentration or patients with colorectal carcinoma (CRC) have also been investigated. The emphasis was made on samples from patients with CRC, a disease accompanied by systemic anemia and increased iron accumulation in cancer cells. CRC was a model system for the analysis of IGFBP-3 internalisation via Tf-TfR pathway. The concentration of IGFBP-3/Tf complexes in healthy adults was measured to be 241 ± 62 μg/L, which makes up to 5-7% of the total IGFBP-3. The results have shown that in impaired iron metabolism, the formation of complexes is highly dependent on the iron concentration, then IGFBP-3 concentration, as well as the concentration of other proteins involved in iron metabolism, such as ferritin. IGFBP-3/Tf complexes isolated form serum..