RESUMO
Successful plant reproduction depends on the adequate development of flower organs controlled by cell proliferation and other processes. The SCI1 gene regulates cell proliferation and affects the final size of the female reproductive organ. To unravel the molecular mechanism exerted by SCI1 in cell proliferation control, we searched for its interaction partners through semi-in vivo pulldown experiments, uncovering a cyclin-dependent kinase, NtCDKG;2. Bimolecular fluorescence complementation (BiFC) and co-localization experiments showed that SCI1 interacts with NtCDKG;2 and its cognate NtCyclin L in nucleoli and splicing speckles. The screening of a yeast two-hybrid (Y2H) cDNA library using SCI1 as bait revealed a novel DEAD-box RNA helicase (NtRH35). The interaction between the NtCDKG;2-NtCyclin L complex, and NtRH35 was also shown. Subcellular localization experiments showed that SCI1, NtRH35, and the NtCDKG;2-NtCyclin L complex associate with each other within splicing speckles. The Y2H screening of NtCDKG;2 and NtRH35 identified the conserved spliceosome components U2a', NKAP, and CACTIN. This work presents SCI1 and its interactors NtCDKG;2-NtCyclin L complex, and NtRH35 as new spliceosome-associated proteins. Our findings reveal a network of interactions and suggest that SCI1 may regulate cell proliferation through the splicing process. This study provides new valuable insights into the intricate molecular pathways governing plant development.
RESUMO
Septins are cytoskeletal proteins and their interaction with membranes is crucial for their role in various cellular processes. Septins have polybasic regions (PB1 and PB2) which are important for lipid interaction. Earlier, we and others have highlighted the role of the septin C-terminal domain (CTD) to membrane interaction. However, detailed information on residues/group of residues important for such feature is lacking. In this study, we investigate the lipid-binding profile of Schistosoma mansoni Septin10 (SmSEPT10) using PIP strip and Langmuir monolayer adsorption assays. Our findings highlight the CTD as the primary domain responsible for lipid interaction in SmSEPT10, showing binding to phosphatidylinositol phosphates. SmSEPT10 CTD contains a conserved polybasic region (PB3) present in both animals and fungi septins, and a Lys (K367) within its putative amphipathic helix (AH) that we demonstrate as important for lipid binding. PB3 deletion or mutation of this Lys (K367A) strongly impairs lipid interaction. Remarkably, we observe that the AH within a construct lacking the final 43 amino acid residues is insufficient for lipid binding. Furthermore, we investigate the homocomplex formed by SmSEPT10 CTD in solution by cross-linking experiments, CD spectroscopy, SEC-MALS and SEC-SAXS. Taken together, our studies define the lipid-binding region in SmSEPT10 and offer insights into the molecular basis of septin-membrane binding. This information is particularly relevant for less-studied non-human septins, such as SmSEPT10.
Assuntos
Schistosoma mansoni , Septinas , Schistosoma mansoni/genética , Schistosoma mansoni/metabolismo , Septinas/metabolismo , Septinas/química , Septinas/genética , Animais , Ligação Proteica , Domínios Proteicos , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Helminto/química , Proteínas de Helminto/metabolismo , Proteínas de Helminto/genética , Lipídeos/químicaRESUMO
The final shape and size of plant organs are determined by a network of genes that modulate cell proliferation and expansion. Among those, SCI1 (Stigma/style Cell-cycle Inhibitor 1) functions by inhibiting cell proliferation during pistil development. Alterations in SCI1 expression levels can lead to remarkable stigma/style size changes. Recently, we demonstrated that SCI1 starts to be expressed at the specification of the Nicotiana tabacum floral meristem and is expressed at all floral meristematic cells. To elucidate how SCI1 regulates cell proliferation, we screened a stigma/style cDNA library through the yeast two-hybrid (Y2H) system, using SCI1 as bait. Among the interaction partners, we identified the 14-3-3D protein of the Non-Epsilon group. The interaction between SCI1 and 14-3-3D was confirmed by pulldown and co-immunoprecipitation experiments. 14-3-3D forms homo- and heterodimers in the cytoplasm of plant cells and interacts with SCI1 in the nucleus, as demonstrated by Bimolecular Fluorescence Complementation (BiFC). Analyses of SCI1-GFP fluorescence through the cell-cycle progression revealed its presence in the nucleoli during interphase and prophase. At metaphase, SCI1-GFP fluorescence faded and was no longer detected at anaphase, reappearing at telophase. Upon treatment with the 26S proteasome inhibitor MG132, SCI1-GFP was stabilized during cell division. Site-directed mutagenesis of seven serines into alanines in the predicted 14-3-3 binding sites on the SCI1 sequence prevented its degradation during mitosis. Our results demonstrate that SCI1 degradation at the beginning of metaphase is dependent on the phosphorylation of serine residues and on the action of the 26S proteasome. We concluded that SCI1 stability/degradation is cell-cycle regulated, consistent with its role in fine-tuning cell proliferation.
RESUMO
Septins are members of a group of GTP-binding proteins highly conserved in eukaryotes, being linked to diverse cell processes, such as cytokinesis and membrane association. On the other hand, the malfunction of septins is linked to several pathological processes including neurodegeneration and oncogenesis. Septins interact with each other forming heterocomplexes that polymerize in filaments. Two types of interface between septins alternate along the filament: the G-interface (involving the GTP binding sites), and the NC-interface. This work focuses on the physiological G-interface of SEPT2, used in the SEPT6G-SEPT2G heterodimer assembly, to verify the impact of this interaction on the thermostability and amyloid formation. We found that the SEPT6G-SEPT2G moves to an irreversible state with the ability to bind thioflavin-T at high temperatures, suggesting its amyloid-like nature. Noteworthy, this takes place at a higher temperature than the one observed to the single septins, showing greater thermal/structural stability. Taken together, our results show that in the absence of the partners, the septin becomes unstable and susceptible to amyloid aggregation/formation even in physiological temperatures, and the G-interface appears to have a critical role in this process.
Assuntos
Amiloide/química , Agregados Proteicos , Septinas/química , Septinas/metabolismo , Estabilidade Enzimática , Domínios Proteicos , Multimerização Proteica , Estrutura Quaternária de Proteína , Especificidade por Substrato , TemperaturaRESUMO
The molecular mechanisms responsible for protein structural changes in the central nervous system leading to Alzheimer's disease are unknown, but there is evidence that a family of proteins known as septins may be involved. Septins are a conserved group of GTP-binding proteins which participate in various cellular processes, including polarity determination and membrane dynamics. SEPT1, SEPT4, and SEPT2 have been found in deposits known as neurofibrillary tangles and glial fibrils in Alzheimer's disease. In this study, we provide molecular-level information for the interaction of SEPT2 with Langmuir monolayers at the air/water interface, which are used as simplified membrane models. The high surface activity of SEPT2 causes it to adsorb onto distinct types of lipid Langmuir monolayers, namely dipalmitoylphosphatidylcholine and PtdIns(4,5)P2. However, the interaction with PtdIns(4,5)P2 is much stronger, not only leading to a higher adsorption, but also to SEPT2 remaining inserted within the membrane at high surface pressures. Most importantly, in situ polarization-modulated infrared reflection absorption spectroscopy results indicated that the native secondary structure of SEPT2 is preserved upon interacting with PtdIns(4,5)P2, but not when dipalmitoylphosphatidylcholine is at the air/water interface. Taken together, the results presented here suggest that the interaction between SEPT2 and the cell membrane may play an important role in the assembly of SEPT2 into amyloid-like fibers.
Assuntos
Lipídeos de Membrana/química , Membranas Artificiais , Septinas/química , Espectrofotometria Infravermelho/métodos , 1,2-Dipalmitoilfosfatidilcolina/química , Adsorção , Varredura Diferencial de Calorimetria , Cinética , Lipídeos de Membrana/metabolismo , Fosfatidilinositol 4,5-Difosfato/química , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Septinas/metabolismo , Propriedades de SuperfícieRESUMO
Pulchellin is a Ribosome Inactivating Protein containing an A-chain (PAC), whose toxic activity requires crossing the endoplasmic reticulum (ER) membrane. In this paper, we investigate the interaction between recombinant PAC (rPAC) and Langmuir monolayers of dipalmitoyl phosphatidyl glycerol (DPPG), which served as membrane model. Three catalytically active, truncated PACs with increasing deletion of the C-terminal region, possessing 244, 239 and 236 residues (rPAC(244), rPAC(239) and rPAC(236)), were studied. rPAC had the strongest interaction with the DPPG monolayer, inducing a large expansion in its surface pressure-area isotherm. The affinity to DPPG decreased with increased deletion of the C-terminal region. When the C-terminal region was deleted completely (rPAC(236)), the interaction was recovered, probably because other hydrophobic regions were exposed to the membrane. Using Polarization Modulated-Infrared Reflection Absorption Spectroscopy (PM-IRRAS) we observed that at a bare air/water interface rPAC comprised mainly α-helix structures, the C-terminal region had unordered structures when interacting with DPPG. For rPAC(236) the α-helices were preserved even in the presence of DPPG. These results confirm the importance of the C-terminal region for PAC-ER membrane interaction. The partial unfolding only with preserved C-terminal appears a key step for the protein to reach the cytosol and develop its toxic activity.
Assuntos
Proteínas de Plantas/química , Proteínas Inativadoras de Ribossomos/química , Toxinas Biológicas/química , Abrus/química , Sequência de Aminoácidos , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Dados de Sequência Molecular , Fosfatidilgliceróis/química , Fosfatidilgliceróis/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plasmídeos , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Inativadoras de Ribossomos/genética , Proteínas Inativadoras de Ribossomos/metabolismo , Espectrofotometria Infravermelho , Propriedades de Superfície , Toxinas Biológicas/genética , Toxinas Biológicas/metabolismo , Lipossomas Unilamelares/química , Lipossomas Unilamelares/metabolismoRESUMO
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.
Assuntos
Proteínas de Transporte/metabolismo , Septinas/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Ligação Competitiva , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Humanos , Família Multigênica , Ligação Proteica , Septinas/genéticaRESUMO
Three plant proteinase inhibitors BbKI (kallikrein inhibitor) and BbCI (cruzipain inhibitor) from Bauhinia bauhinioides, and a BrTI (trypsin inhibitor) from B. rufa, were examined for other effects in Callosobruchus maculatus development; of these only BrTI affected bruchid emergence. BrTI and BbKI share 81% identities in their primary sequences and the major differences between them are the regions comprising the RGD and RGE motifs in BrTI. These sequences were shown to be essential for BrTI insecticidal activity, since a modified BbKI [that is a recombinant form (BbKIm) with some amino acid residues replaced by those found in BrTI sequence] also strongly inhibited insect development. By using synthetic peptides related to the BrTI sequence, YLEAPVARGDGGLA-NH2 (RGE) and IVYYPDRGETGL-NH2 (RGE), it was found that the peptide with an RGE sequence was able to block normal development of C. maculatus larvae (ED(50) 0.16% and LD(50) 0.09%), this being even more effective than the native protein.
Assuntos
Bauhinia/metabolismo , Besouros/crescimento & desenvolvimento , Inibidores Enzimáticos/metabolismo , Inseticidas/metabolismo , Peptídeo Hidrolases/metabolismo , Doenças das Plantas/genética , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Animais , Bauhinia/química , Bauhinia/genética , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/genética , Inibidores de Cisteína Proteinase/metabolismo , Inibidores Enzimáticos/química , Genes de Plantas , Inseticidas/química , Calicreínas/antagonistas & inibidores , Larva/crescimento & desenvolvimento , Estágios do Ciclo de Vida , Dados de Sequência Molecular , Estrutura Molecular , Peptídeos , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Protozoários , Proteínas Recombinantes , Alinhamento de Sequência , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos , Inibidores da Tripsina/química , Inibidores da Tripsina/genética , Inibidores da Tripsina/metabolismoRESUMO
Diacylglycerol acyltransferase 1 (DGAT1) catalyzes the final and dedicated step in the synthesis of triacylglycerol, which is believed to involve the lipids oleoyl coenzyme A (OCoA) and dioleoyl-sn-glycerol (DOG) as substrates. In this work we investigated the interaction of a specific peptide, referred to as SIT2, on the C-terminal of DGAT1 (HKWCIRHFYKP) with model membranes made with OCoA and DOG in Langmuir monolayers and liposomes. According to the circular dichroism and fluorescence data, conformational changes on SIT2 were seen only on liposomes containing OCoA and DOG. In Langmuir monolayers, SIT2 causes the isotherms of neat OCoA and DOG monolayers to be expanded, but has negligible effect on mixed monolayers of OCoA and DOG. This synergistic interaction between SIT2 and DOG+OCoA may be rationalized in terms of a molecular model in which SIT2 may serve as a linkage between the two lipids. Our results therefore provide molecular-level evidence for the interaction between this domain and the substrates OCoA and DOG for the synthesis of triacylglycerol.