Functional and structural studies of human SEPT2: determinant factors triggering the sefl-assembly into amyloid fibrils

As septinas fazem parte de uma família de proteínas de ligação ao nucleotídeo guanina. As septinas têm mostrado ter um papel importante na citocinese e outros processos celulares, incluindo a determinação da polaridade celular e reorganização do citoesqueleto. Todos os membros da família de septinas são compostos por três domínios: um N-terminal variável, um domínio central GTPase e uma região C-terminal que inclui sequências de coiled-coil. Septinas possuem uma característica de polimerizarem para formar complexos hetero-oligoméricos altamente organizados, in vivo e in vitro. Estruturas homo-oligoméricas também foram observadas, embora sua função ainda não esteja bem estabelecida. A Septina 2 humana (SEPT2) se acumula no sulco de clivagem de células em divisão, desde a anáfase até a telófase, além de interagir com a actina, e também está envolvida em doenças neurodegenerativas, como mal de Azheimer. Nesse estudo, a ORF que codifica SEPT2, bem como os fragmentos que codificam seus domínios, foram clonados, expressos em E.coli e purificados por cromatografia de afinidade e cromatografia de exclusão molecular. Os produtos foram analisados por espectroscopia de dicroísmo circular, espalhamento de luz a ângulo fixo e espectroscopia de fluorescência extrínseca, usando Tioflavina-T, que é um marcador clássico para fibras amilóides. Em todos os casos, os produtos formaram homodímeros in vitro, e também agregaram em temperaturas fisiológicas. O desenovelamento térmico das proteínas recombinantes revelou a presença de uma população intermediária de desenovelamento, rica em folhas-β, e que ligam Tioflavina-T, sugerindo uma estrutura amiloidogênica para essa proteína, confirmada pelos programas de predição TANGO e WALTZ. Imagens dessas fibras foram obtidas usando Microscopia eletrônica de Transmissão, evidenciando uma agregação organizada das proteínas. Além disso, usando monocamadas de Langmuir, foi possível confirmar a ligação específica de SEPT2 ao fosfolipídeo fosfatidilinositol 4,5-bifosfato (PtdIns(4,5)P2). Essa ligação específica mantém a estrutura secundária de SEPT2, observada pela técnica PM-IRRAS, algo que não ocorre caso o lipídio seja inespecífico, sugerindo uma associação de SEPT2 com a membrana plasmática e podendo ter um papel na regulação das septinas. Por meio da técnica de duplo híbrido em levedura, identificamos proteínas que interagem com a SEPT2, como a MPBI e a DCTN2, auxiliando na elucidação de processos em que a SEPT2 possa participar. O conjunto dos resultados sobre a estabilidade, os processos de agregação de SEPT2 e a identificação de novos parceiros protéicos de interação, obtidos nesse trabalho, contribuíram para o melhor entendimento da função da SEPT2 e de seu envolvimento em desordens neurodegerenativas.Septins are members of a conserved group of GTP-binding and filament-forming proteins. They are involved in a variety of cellular processes, such as microtubule regulation, vesicle trafficking, the formation of scaffolding platforms and actin dynamics. Human Septin 2 (SEPT2) has an N-terminal polybasic region responsible for lipid binding, a GTPase domain, and a C-terminal domain. SEPT2 is essential for cytokinesis and it is found in many tissues, mainly in the brain. Together with SEPT1 and SEPT4, it is accumulated in deposits known as neurofibrillary tangles in Alzheimers disease, which is evidence that SEPT2 may be involved in this process. In this study, the human SEPT2, and its domains, were cloned, expressed in E.coli and purified by affinity and size-exclusion chromatographies. The proteins form homodimers in vitro, suggesting that the GTPase domain is enough to promote the oligomerization. Thermal unfolding revealed the formation of aggregates under physiological conditions, which have the ability to bind a specific amyloid dye, Thioflavin-T, suggesting them to be an amyloidal fiber. Besides, in silico prediction programs, TANGO and WALTZ, corroborate that SEPT2 contain regions with high probability of aggregation and amyloidogenic formation, respectively. Moreover, we observed 20-50 nm thick filamentous structures by electron microscopy of negatively stained. Using Langmuir monolayers at the cell membrane lipid packing, SEPT2 and SEPT2NG bound to the phospholipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). Results from in situ PM-IRRAS experiments indicated that the secondary structure of SEPT2 is preserved upon interacting with PtdIns(4,5)P2, but not when interacting with DPPC - which is not specific for SEPT2 - at the air/water interface suggesting an association with the plasma membrane and a role in septin regulation. Furthermore, we also identified protein partners of SEPT2, from both human leukocyte and brain fetal cDNA libraries, using the yeast two-hybrid system. SEPT2 was shown to interact with: septins 6 and 4; a serine-protease and a MAP inhibitory protein; an ubiquitin-conjugating enzyme; and proteins related to cellular division. Thus, taken together this study contributed for the knowledgment of the stability and the aggregation kinetic of the SEPT2, leading to a better understanding of this protein and their role in neurodegenerative disorders

A Draft of the Human Septin Interactome

Background: Septins belong to the GTPase superclass of proteins and have been functionally implicated in cytokinesis and the maintenance of cellular morphology. They are found in all eukaryotes, except in plants. In mammals, 14 septins have been described that can be divided into four groups. It has been shown that mammalian septins can engage in homo- and heterooligomeric assemblies, in the form of filaments, which have as a basic unit a hetero-trimeric core. In addition, it has been speculated that the septin filaments may serve as scaffolds for the recruitment of additional proteins. Methodology/Principal Findings: Here, we performed yeast two-hybrid screens with human septins 1-10, which include representatives of all four septin groups. Among the interactors detected, we found predominantly other septins, confirming the tendency of septins to engage in the formation of homo- and heteropolymeric filaments. Conclusions/Significance: If we take as reference the reported arrangement of the septins 2, 6 and 7 within the heterofilament, (7-6-2-2-6-7), we note that the majority of the observed interactions respect the ""group rule"", i.e. members of the same group (e. g. 6, 8, 10 and 11) can replace each other in the specific position along the heterofilament. Septins of the SEPT6 group preferentially interacted with septins of the SEPT2 group (p<0.001), SEPT3 group (p<0.001) and SEPT7 group (p<0.001). SEPT2 type septins preferentially interacted with septins of the SEPT6 group (p<0.001) aside from being the only septin group which interacted with members of its own group. Finally, septins of the SEPT3 group interacted preferentially with septins of the SEPT7 group (p<0.001). Furthermore, we found non-septin interactors which can be functionally attributed to a variety of different cellular activities, including: ubiquitin/sumoylation cycles, microtubular transport and motor activities, cell division and the cell cycle, cell motility, protein phosphorylation/signaling, endocytosis, and apoptosis.Fundao de Amparo a Pesquisa do Estado Sao Paulo (Fapesp)CAPES: Coordenao de Aperfeioamento de Pessoal de Navel SuperiorConselho Nacional de Pesquisa e Desenvolvimento (CNPq)Laboratorio Nacional de Biociencias-Centro Nacional de Pesquisa em Energia e Materais (LNBio-CNPEM

Self assembly of human septin 2 into amyloid filaments

Septins are a conserved group of GTP-binding proteins that form hetero-oligomeric complexes which assemble into filaments. These are essential for septin function, including their role in cytokinesis, cell division, exocytosis and membrane trafficking. Septin 2 (SEPT2) is a member of the septin family and has been associated with neurofibrillary tangles and other pathological features of senile plaques in Alzheimer's disease. An in silico analysis of the amino acid sequence of SEPT2 identified regions with a significant tendency to aggregate and/or form amyloid. These were all observed within the GTP-binding domain. This was consistent with the experimental identification of a structure rich in beta-sheet during temperature induced unfolding transitions observed for both the full length protein and the GTP-binding domain alone. This intermediate state is characterized by irreversible aggregation and has the ability to bind Thioflavin-T, suggesting its amyloid nature. Under electron microscopy, fibers extending for several micrometers in length could be visualized. The results shown in this study support the hypothesis that single septins, when present in excess or with unbalanced stoichiometries, may be unstable and assemble into amyloid-like structures. (C) 2011 Elsevier Masson SAS. All rights reserved.FAPESPFAPES

Septin C-Terminal Domain Interactions: Implications for Filament Stability and Assembly

Septins form a conserved family of filament forming GTP binding proteins found in a wide range of eukaryotic cells. They share a common structural architecture consisting of an N-terminal domain, a central GTP binding domain and a C-terminal domain, which is often predicted to adopt a coiled-coil conformation, at least in part. The crystal structure of the human SEPT2/SEPT6/SEPT7 heterocomplex has revealed the importance of the GTP binding domain in filament formation, but surprisingly no electron density was observed for the C-terminal domains and their function remains obscure. The dearth of structural information concerning the C-terminal region has motivated the present study in which the putative C-terminal domains of human SEPT2, SEPT6 and SEPT7 were expressed in E. coli and purified to homogeneity. The thermal stability and secondary structure content of the domains were studied by circular dichroism spectroscopy, and homo- and hetero-interactions were investigated by size exclusion chromatography, chemical cross-linking, analytical ultracentrifugation and surface plasmon resonance. Our results show that SEPT6-C and SEPT7-C are able to form both homo- and heterodimers with a high alpha-helical content in solution. The heterodimer is elongated and considerably more stable than the homodimers, with a K (D) of 15.8 nM. On the other hand, the homodimer SEPT2-C has a much lower affinity, with a K (D) of 4 mu M, and a moderate alpha-helical content. Our findings present the first direct experimental evidence toward better understanding the biophysical properties and coiled-coil pairings of such domains and their potential role in filament assembly and stability.FAPESP (via its CEPID)CSIC-CNPqMCINN [BFU2008-00013]CAPE