RESUMO
Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of familial Parkinson's disease. LRRK2 is a multi-domain protein containing a kinase and GTPase. Using correlative light and electron microscopy, in situ cryo-electron tomography, and subtomogram analysis, we reveal a 14-Å structure of LRRK2 bearing a pathogenic mutation that oligomerizes as a right-handed double helix around microtubules, which are left-handed. Using integrative modeling, we determine the architecture of LRRK2, showing that the GTPase and kinase are in close proximity, with the GTPase closer to the microtubule surface, whereas the kinase is exposed to the cytoplasm. We identify two oligomerization interfaces mediated by non-catalytic domains. Mutation of one of these abolishes LRRK2 microtubule-association. Our work demonstrates the power of cryo-electron tomography to generate models of previously unsolved structures in their cellular environment.
Assuntos
Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/química , Microtúbulos/metabolismo , Doença de Parkinson/metabolismo , Citoplasma/metabolismo , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , Células HEK293 , Humanos , Microscopia Eletrônica de Transmissão , Microtúbulos/química , Modelos Químicos , Mutação , Doença de Parkinson/genética , Doença de Parkinson/patologia , Fosfotransferases/química , Fosfotransferases/metabolismo , Domínios Proteicos , Repetições WD40RESUMO
Dominant optic atrophy is one of the leading causes of childhood blindness. Around 60-80% of cases1 are caused by mutations of the gene that encodes optic atrophy protein 1 (OPA1), a protein that has a key role in inner mitochondrial membrane fusion and remodelling of cristae and is crucial for the dynamic organization and regulation of mitochondria2. Mutations in OPA1 result in the dysregulation of the GTPase-mediated fusion process of the mitochondrial inner and outer membranes3. Here we used cryo-electron microscopy methods to solve helical structures of OPA1 assembled on lipid membrane tubes, in the presence and absence of nucleotide. These helical assemblies organize into densely packed protein rungs with minimal inter-rung connectivity, and exhibit nucleotide-dependent dimerization of the GTPase domains-a hallmark of the dynamin superfamily of proteins4. OPA1 also contains several unique secondary structures in the paddle domain that strengthen its membrane association, including membrane-inserting helices. The structural features identified in this study shed light on the effects of pathogenic point mutations on protein folding, inter-protein assembly and membrane interactions. Furthermore, mutations that disrupt the assembly interfaces and membrane binding of OPA1 cause mitochondrial fragmentation in cell-based assays, providing evidence of the biological relevance of these interactions.
Assuntos
Microscopia Crioeletrônica , GTP Fosfo-Hidrolases , Mitocôndrias , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , GTP Fosfo-Hidrolases/ultraestrutura , Fusão de Membrana , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Dinâmica Mitocondrial , Membranas Mitocondriais/metabolismo , Mutação , Nucleotídeos/metabolismo , Ligação Proteica/genética , Domínios Proteicos , Dobramento de Proteína , Multimerização Proteica , Estrutura Secundária de Proteína , HumanosRESUMO
The SEA complex (SEAC) is a growth regulator that acts as a GTPase-activating protein (GAP) towards Gtr1, a Rag GTPase that relays nutrient status to the Target of Rapamycin Complex 1 (TORC1) in yeast1. Functionally, the SEAC has been divided into two subcomplexes: SEACIT, which has GAP activity and inhibits TORC1, and SEACAT, which regulates SEACIT2. This system is conserved in mammals: the GATOR complex, consisting of GATOR1 (SEACIT) and GATOR2 (SEACAT), transmits amino acid3 and glucose4 signals to mTORC1. Despite its importance, the structure of SEAC/GATOR, and thus molecular understanding of its function, is lacking. Here, we solve the cryo-EM structure of the native eight-subunit SEAC. The SEAC has a modular structure in which a COPII-like cage corresponding to SEACAT binds two flexible wings, which correspond to SEACIT. The wings are tethered to the core via Sea3, which forms part of both modules. The GAP mechanism of GATOR1 is conserved in SEACIT, and GAP activity is unaffected by SEACAT in vitro. In vivo, the wings are essential for recruitment of the SEAC to the vacuole, primarily via the EGO complex. Our results indicate that rather than being a direct inhibitor of SEACIT, SEACAT acts as a scaffold for the binding of TORC1 regulators.
Assuntos
Microscopia Crioeletrônica , Proteínas Ativadoras de GTPase , Complexos Multienzimáticos , Animais , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , GTP Fosfo-Hidrolases/ultraestrutura , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/metabolismo , Proteínas Ativadoras de GTPase/ultraestrutura , Mamíferos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Complexos Multienzimáticos/ultraestrutura , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Aminoácidos , Glucose , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/química , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismoRESUMO
In contrast to the bacterial translation machinery, mitoribosomes and mitochondrial translation factors are highly divergent in terms of composition and architecture. There is increasing evidence that the biogenesis of mitoribosomes is an intricate pathway, involving many assembly factors. To better understand this process, we investigated native assembly intermediates of the mitoribosomal large subunit from the human parasite Trypanosoma brucei using cryo-electron microscopy. We identify 28 assembly factors, 6 of which are homologous to bacterial and eukaryotic ribosome assembly factors. They interact with the partially folded rRNA by specifically recognizing functionally important regions such as the peptidyltransferase center. The architectural and compositional comparison of the assembly intermediates indicates a stepwise modular assembly process, during which the rRNA folds toward its mature state. During the process, several conserved GTPases and a helicase form highly intertwined interaction networks that stabilize distinct assembly intermediates. The presented structures provide general insights into mitoribosomal maturation.
Assuntos
Ribossomos Mitocondriais/química , RNA Ribossômico/metabolismo , Subunidades Ribossômicas Maiores/química , Trypanosoma brucei brucei/metabolismo , Microscopia Crioeletrônica , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/metabolismo , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Ribossomos Mitocondriais/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , RNA Ribossômico/química , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Maiores/metabolismo , Trypanosoma brucei brucei/genéticaRESUMO
RAS GTPases associate with the biological membrane where they function as molecular switches to regulate cell growth. Recent studies indicate that RAS proteins oligomerize on membranes, and disrupting these assemblies represents an alternative therapeutic strategy. However, conflicting reports on RAS assemblies, ranging in size from dimers to nanoclusters, have brought to the fore key questions regarding the stoichiometry and parameters that influence oligomerization. Here, we probe three isoforms of RAS [Kirsten Rat Sarcoma viral oncogene (KRAS), Harvey Rat Sarcoma viral oncogene (HRAS), and Neuroblastoma oncogene (NRAS)] directly from membranes using mass spectrometry. We show that KRAS on membranes in the inactive state (GDP-bound) is monomeric but forms dimers in the active state (GTP-bound). We demonstrate that the small molecule BI2852 can induce dimerization of KRAS, whereas the binding of effector proteins disrupts dimerization. We also show that RAS dimerization is dependent on lipid composition and reveal that oligomerization of NRAS is regulated by palmitoylation. By monitoring the intrinsic GTPase activity of RAS, we capture the emergence of a dimer containing either mixed nucleotides or GDP on membranes. We find that the interaction of RAS with the catalytic domain of Son of Sevenless (SOScat) is influenced by membrane composition. We also capture the activation and monomer to dimer conversion of KRAS by SOScat. These results not only reveal the stoichiometry of RAS assemblies on membranes but also uncover the impact of critical factors on oligomerization, encompassing regulation by nucleotides, lipids, and palmitoylation.
Assuntos
Membrana Celular , Multimerização Proteica , Proteínas Proto-Oncogênicas p21(ras) , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/química , Humanos , Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , GTP Fosfo-Hidrolases/metabolismo , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/genética , Lipoilação , Proteínas ras/metabolismo , Proteínas ras/química , Guanosina Trifosfato/metabolismo , Guanosina Difosfato/metabolismoRESUMO
FtsZ, the tubulin homolog essential for bacterial cell division, assembles as the Z-ring at the division site, and directs peptidoglycan synthesis by treadmilling. It is unclear how FtsZ achieves kinetic polarity that drives treadmilling. To obtain insights into fundamental features of FtsZ assembly dynamics independent of peptidoglycan synthesis, we carried out structural and biochemical characterization of FtsZ from the cell wall-less bacteria, Spiroplasma melliferum (SmFtsZ). Interestingly the structures of SmFtsZ, bound to GDP and GMPPNP respectively, were captured as domain swapped dimers. SmFtsZ was found to be a slower GTPase with a higher critical concentration (CC) compared to Escherichia coli FtsZ (EcFtsZ). In FtsZs, a conformational switch from R-state (close) to T-state (open) favors polymerization. We identified that Phe224, located at the interdomain cleft of SmFtsZ, is crucial for R- to T-state transition. SmFtsZF224M exhibited higher GTPase activity and lower CC, whereas the corresponding EcFtsZM225F resulted in cell division defects in E. coli. Our results demonstrate that relative rotation of the domains is a rate-limiting step of polymerization. Our structural analysis suggests that the rotation is plausibly triggered upon addition of a GTP-bound monomer to the filament through interaction of the preformed N-terminal domain (NTD). Hence, addition of monomers to the NTD-exposed end of filament is slower in comparison to the C-terminal domain (CTD) end, thus explaining kinetic polarity. In summary, the study highlights the importance of interdomain interactions and conformational changes in regulating FtsZ assembly dynamics.
Assuntos
Proteínas de Bactérias , Proteínas do Citoesqueleto , Escherichia coli , Proteínas do Citoesqueleto/metabolismo , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Escherichia coli/metabolismo , Escherichia coli/genética , Domínios Proteicos , Multimerização Proteica , Guanosina Difosfato/metabolismo , Guanosina Difosfato/química , GTP Fosfo-Hidrolases/metabolismo , GTP Fosfo-Hidrolases/química , Divisão CelularRESUMO
Several ubiquitin chain types have remained unstudied, mainly because tools and techniques to detect these posttranslational modifications are scarce. Linkage-specific antibodies have shaped our understanding of the roles and dynamics of polyubiquitin signals but are available for only five out of eight linkage types. We here characterize K6- and K33-linkage-specific "affimer" reagents as high-affinity ubiquitin interactors. Crystal structures of affimers bound to their cognate chain types reveal mechanisms of specificity and a K11 cross-reactivity in the K33 affimer. Structure-guided improvements yield superior affinity reagents suitable for western blotting, confocal fluorescence microscopy and pull-down applications. This allowed us to identify RNF144A and RNF144B as E3 ligases that assemble K6-, K11-, and K48-linked polyubiquitin in vitro. A protocol to enrich K6-ubiquitinated proteins from cells identifies HUWE1 as a main E3 ligase for this chain type, and we show that mitofusin-2 is modified with K6-linked polyubiquitin in a HUWE1-dependent manner.
Assuntos
GTP Fosfo-Hidrolases/química , Proteínas Mitocondriais/química , Sondas Moleculares/química , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Ubiquitina-Proteína Ligases/química , Ubiquitina/química , Motivos de Aminoácidos , Sítios de Ligação , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Cinética , Lisina/química , Lisina/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Proteínas Supressoras de Tumor , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
The formation of complexes between Rab11 and its effectors regulates multiple aspects of membrane trafficking, including recycling and ciliogenesis. WD repeat-containing protein 44 (WDR44) is a structurally uncharacterized Rab11 effector that regulates ciliogenesis by competing with prociliogenesis factors for Rab11 binding. Here, we present a detailed biochemical and biophysical characterization of the WDR44-Rab11 complex and define specific residues mediating binding. Using AlphaFold2 modeling and hydrogen/deuterium exchange mass spectrometry, we generated a molecular model of the Rab11-WDR44 complex. The Rab11-binding domain of WDR44 interacts with switch I, switch II, and the interswitch region of Rab11. Extensive mutagenesis of evolutionarily conserved residues in WDR44 at the interface identified numerous complex-disrupting mutations. Using hydrogen/deuterium exchange mass spectrometry, we found that the dynamics of the WDR44-Rab11 interface are distinct from the Rab11 effector FIP3, with WDR44 forming a more extensive interface with the switch II helix of Rab11 compared with FIP3. The WDR44 interaction was specific to Rab11 over evolutionarily similar Rabs, with mutations defining the molecular basis of Rab11 specificity. Finally, WDR44 can be phosphorylated by Sgk3, with this leading to reorganization of the Rab11-binding surface on WDR44. Overall, our results provide molecular detail on how WDR44 interacts with Rab11 and how Rab11 can form distinct effector complexes that regulate membrane trafficking events.
Assuntos
GTP Fosfo-Hidrolases , Quinase I-kappa B , Modelos Moleculares , Proteínas rab de Ligação ao GTP , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , Quinase I-kappa B/metabolismo , Ligação Proteica , Proteínas rab de Ligação ao GTP/química , Proteínas rab de Ligação ao GTP/metabolismo , Espectrometria de MassasRESUMO
Long noncoding RNAs (lncRNAs) are emerging as essential players in multiple biological processes. Mitochondrial dynamics, comprising the continuous cycle of fission and fusion, are required for healthy mitochondria that function properly. Despite long-term recognition of its significance in cell-fate control, the mechanism underlying mitochondrial fusion is not completely understood, particularly regarding the involvement of lncRNAs. Here, we show that the lncRNA HITT (HIF-1α inhibitor at translation level) can specifically localize in mitochondria. Cells expressing higher levels of HITT contain fragmented mitochondria. Conversely, we show that HITT knockdown cells have more tubular mitochondria than is present in control cells. Mechanistically, we demonstrate HITT directly binds mitofusin-2 (MFN2), a core component that mediates mitochondrial outer membrane fusion, by the in vitro RNA pull-down and UV-cross-linking RNA-IP assays. In doing so, we found HITT disturbs MFN2 homotypic or heterotypic complex formation, attenuating mitochondrial fusion. Under stress conditions, such as ultraviolet radiation, we in addition show HITT stability increases as a consequence of MiR-205 downregulation, inhibiting MFN2-mediated fusion and leading to apoptosis. Overall, our data provide significant insights into the roles of organelle (mitochondria)-specific resident lncRNAs in regulating mitochondrial fusion and also reveal how such a mechanism controls cellular sensitivity to UV radiation-induced apoptosis.
Assuntos
GTP Fosfo-Hidrolases , Mitocôndrias , Dinâmica Mitocondrial , Proteínas Mitocondriais , Complexos Multiproteicos , RNA Longo não Codificante , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/efeitos da radiação , Dinâmica Mitocondrial/efeitos da radiação , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Raios Ultravioleta , MicroRNAs/metabolismo , Apoptose/efeitos da radiação , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Membranas Mitocondriais/metabolismoRESUMO
Many GTPases have been shown to utilize ATP too as the phosphoryl donor. Both GTP and ATP are important molecules in the cellular environments and play multiple and discrete functional role within the cells. In our present study, we showed that one of the purine metabolic enzymes Adenylosuccinate synthetase from Leishmania donovani (LdAdSS) which belongs to the BioD-superfamily of GTPases can also carry out the catalysis by hydrolysing ATP instead of its cognate substrate GTP albeit with less efficiency. Biochemical and biophysical studies indicated its ability to bind to ATP too but at a higher concentration of ATP compared to that of GTP. Sequence analysis and molecular dynamic simulations suggested that residues of the switch loop and the G4-G5 (593SAXD596) connected motif of LdAdSS plays a role in determining the nucleotide specificity. Though the crucial interaction between Asp596 and the nucleotide is broken when ATP is bound, interactions between the Ala594 and the adenine ring of ATP could still hold ATP in the GTP binding site. The results of the present study suggested that though LdAdSS is GTP specific, it still shows ATP hydrolysing activity.
Assuntos
Trifosfato de Adenosina , Adenilossuccinato Sintase , Guanosina Trifosfato , Leishmania donovani , Leishmania donovani/enzimologia , Leishmania donovani/metabolismo , Leishmania donovani/genética , Trifosfato de Adenosina/metabolismo , Guanosina Trifosfato/metabolismo , Adenilossuccinato Sintase/metabolismo , Adenilossuccinato Sintase/química , Especificidade por Substrato , Simulação de Dinâmica Molecular , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , GTP Fosfo-Hidrolases/metabolismo , GTP Fosfo-Hidrolases/químicaRESUMO
Dynamin, the founding member of a family of dynamin-like proteins (DLPs) implicated in membrane remodelling, has a critical role in endocytic membrane fission events. The use of complementary approaches, including live-cell imaging, cell-free studies, X-ray crystallography and genetic studies in mice, has greatly advanced our understanding of the mechanisms by which dynamin acts, its essential roles in cell physiology and the specific function of different dynamin isoforms. In addition, several connections between dynamin and human disease have also emerged, highlighting specific contributions of this GTPase to the physiology of different tissues.
Assuntos
Membrana Celular/fisiologia , Dinaminas/fisiologia , Animais , Membrana Celular/metabolismo , Dinaminas/química , Dinaminas/genética , Dinaminas/metabolismo , Endocitose/genética , Endocitose/fisiologia , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , GTP Fosfo-Hidrolases/fisiologia , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Isoenzimas/fisiologia , Mamíferos/genética , Mamíferos/metabolismo , Fluidez de Membrana/genética , Camundongos , Modelos Biológicos , Modelos Moleculares , Conformação ProteicaRESUMO
Mitochondrial inheritance, genome maintenance and metabolic adaptation depend on organelle fission by dynamin-related protein 1 (DRP1) and its mitochondrial receptors. DRP1 receptors include the paralogues mitochondrial dynamics proteins of 49 and 51 kDa (MID49 and MID51) and mitochondrial fission factor (MFF); however, the mechanisms by which these proteins recruit and regulate DRP1 are unknown. Here we present a cryo-electron microscopy structure of full-length human DRP1 co-assembled with MID49 and an analysis of structure- and disease-based mutations. We report that GTP induces a marked elongation and rotation of the GTPase domain, bundle-signalling element and connecting hinge loops of DRP1. In this conformation, a network of multivalent interactions promotes the polymerization of a linear DRP1 filament with MID49 or MID51. After co-assembly, GTP hydrolysis and exchange lead to MID receptor dissociation, filament shortening and curling of DRP1 oligomers into constricted and closed rings. Together, these views of full-length, receptor- and nucleotide-bound conformations reveal how DRP1 performs mechanical work through nucleotide-driven allostery.
Assuntos
Proteínas Quinases Associadas com Morte Celular/metabolismo , Proteínas Quinases Associadas com Morte Celular/ultraestrutura , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/ultraestrutura , Fatores de Alongamento de Peptídeos/metabolismo , Fatores de Alongamento de Peptídeos/ultraestrutura , Regulação Alostérica , Sítios de Ligação/genética , Microscopia Crioeletrônica , Proteínas Quinases Associadas com Morte Celular/química , Proteínas Quinases Associadas com Morte Celular/genética , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , GTP Fosfo-Hidrolases/ultraestrutura , Guanosina Trifosfato/metabolismo , Humanos , Hidrólise , Proteínas Mitocondriais/química , Modelos Moleculares , Mutação , Fatores de Alongamento de Peptídeos/química , Fosforilação , Domínios Proteicos , Rotação , Relação Estrutura-AtividadeRESUMO
Aberrant Ras signaling is linked to a wide spectrum of hyperproliferative diseases, and components of the signaling pathway, including Ras, have been the subject of intense and ongoing drug discovery efforts. The cellular activity of Ras is modulated by its association with the guanine nucleotide exchange factor Son of sevenless (Sos), and the high-resolution crystal structure of the Ras-Sos complex provides a basis for the rational design of orthosteric Ras ligands. We constructed a synthetic Sos protein mimic that engages the wild-type and oncogenic forms of nucleotide-bound Ras and modulates downstream kinase signaling. The Sos mimic was designed to capture the conformation of the Sos helix-loop-helix motif that makes critical contacts with Ras in its switch region. Chemoproteomic studies illustrate that the proteomimetic engages Ras and other cellular GTPases. The synthetic proteomimetic resists proteolytic degradation and enters cells through macropinocytosis. As such, it is selectively toxic to cancer cells with up-regulated macropinocytosis, including those that feature oncogenic Ras mutations.
Assuntos
Complexos Multiproteicos/ultraestrutura , Conformação Proteica , Proteína Son Of Sevenless de Drosófila/ultraestrutura , Proteínas ras/ultraestrutura , Animais , Biomimética , Cristalografia por Raios X , Descoberta de Drogas , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/ultraestrutura , Células HCT116 , Sequências Hélice-Alça-Hélice/genética , Humanos , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Proteoma/genética , Transdução de Sinais/genética , Proteína Son Of Sevenless de Drosófila/química , Proteína Son Of Sevenless de Drosófila/genética , Proteínas ras/química , Proteínas ras/genéticaRESUMO
Mitochondria are double-membraned organelles with variable shapes influenced by metabolic conditions, developmental stage, and environmental stimuli. Their dynamic morphology is a result of regulated and balanced fusion and fission processes. Fusion is crucial for the health and physiological functions of mitochondria, including complementation of damaged mitochondrial DNAs and the maintenance of membrane potential. Mitofusins are dynamin-related GTPases that are essential for mitochondrial fusion. They are embedded in the mitochondrial outer membrane and thought to fuse adjacent mitochondria via combined oligomerization and GTP hydrolysis. However, the molecular mechanisms of this process remain unknown. Here we present crystal structures of engineered human MFN1 containing the GTPase domain and a helical domain during different stages of GTP hydrolysis. The helical domain is composed of elements from widely dispersed sequence regions of MFN1 and resembles the 'neck' of the bacterial dynamin-like protein. The structures reveal unique features of its catalytic machinery and explain how GTP binding induces conformational changes to promote GTPase domain dimerization in the transition state. Disruption of GTPase domain dimerization abolishes the fusogenic activity of MFN1. Moreover, a conserved aspartate residue trigger was found to affect mitochondrial elongation in MFN1, probably through a GTP-loading-dependent domain rearrangement. Thus, we propose a mechanistic model for MFN1-mediated mitochondrial tethering, and our results shed light on the molecular basis of mitochondrial fusion and mitofusin-related human neuromuscular disorders.
Assuntos
GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , Guanosina Trifosfato/metabolismo , Mitocôndrias/química , Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Proteínas de Transporte da Membrana Mitocondrial/química , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Sequência de Aminoácidos , Biocatálise , Cristalografia por Raios X , GTP Fosfo-Hidrolases/genética , Humanos , Hidrólise , Fusão de Membrana , Potenciais da Membrana , Proteínas de Transporte da Membrana Mitocondrial/genética , Membranas Mitocondriais/química , Membranas Mitocondriais/metabolismo , Modelos Moleculares , Domínios Proteicos , Multimerização Proteica , Triptofano/metabolismoRESUMO
Controlled changes in mitochondrial morphology participate in cellular signaling cascades. However, the molecular mechanisms modifying mitochondrial shape are largely unknown. Here we show that the mitogen-activated protein (MAP) kinase cascade member extracellular-signal-regulated kinase (ERK) phosphorylates the pro-fusion protein mitofusin (MFN) 1, modulating its participation in apoptosis and mitochondrial fusion. Phosphoproteomic and biochemical analyses revealed that MFN1 is phosphorylated at an atypical ERK site in its heptad repeat (HR) 1 domain. This site proved essential to mediate MFN1-dependent mitochondrial elongation and apoptosis regulation by the MEK/ERK cascade. A mutant mimicking constitutive MFN1 phosphorylation was less efficient in oligomerizing and mitochondria tethering but bound more avidly to the proapoptotic BCL-2 family member BAK, facilitating its activation and cell death. Moreover, neuronal apoptosis following oxygen glucose deprivation and MEK/ERK activation required an intact MFN1(T562). Our data identify MFN1 as an ERK target to modulate mitochondrial shape and apoptosis.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas de Membrana/metabolismo , Mitocôndrias/fisiologia , Proteínas Mitocondriais/metabolismo , Animais , Apoptose , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/metabolismo , GTP Fosfo-Hidrolases/química , Proteínas de Membrana/química , Camundongos , Dinâmica Mitocondrial , Proteínas Mitocondriais/química , Neurônios/citologia , Neurônios/metabolismo , Estresse Oxidativo , Fosforilação , Proteômica , Ratos , Ratos WistarRESUMO
Crosslinking and immunoprecipitation (CLIP) methods are powerful techniques to interrogate direct protein-RNA interactions and dissect posttranscriptional gene regulatory networks. One widely used CLIP variant is photoactivatable ribonucleoside enhanced CLIP (PAR-CLIP) that involves in vivo labeling of nascent RNAs with the photoreactive nucleosides 4-thiouridine (4SU) or 6-thioguanosine (6SG), which can efficiently crosslink to interacting proteins using UVA and UVB light. Crosslinking of 4SU or 6SG to interacting amino acids changes their base-pairing properties and results in characteristic mutations in cDNA libraries prepared for high-throughput sequencing, which can be computationally exploited to remove abundant background from non-crosslinked sequences and help pinpoint RNA binding protein binding sites at nucleotide resolution on a transcriptome-wide scale. Here we present a streamlined protocol for fluorescence-based PAR-CLIP (fPAR-CLIP) that eliminates the need to use radioactivity. It is based on direct ligation of a fluorescently labeled adapter to the 3'end of crosslinked RNA on immobilized ribonucleoproteins, followed by isolation of the adapter-ligated RNA and efficient conversion into cDNA without the previously needed size fractionation on denaturing polyacrylamide gels. These improvements cut the experimentation by half to 2 days and increases sensitivity by 10-100-fold.
Assuntos
DNA Complementar/genética , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Sítios de Ligação , Linhagem Celular , Reagentes de Ligações Cruzadas/química , Eletroforese em Gel de Poliacrilamida , GTP Fosfo-Hidrolases/química , Biblioteca Gênica , Humanos , Imunoprecipitação , Oligonucleotídeos , Reação em Cadeia da Polimerase/métodos , Ligação Proteica , RNA/química , Ribonucleoproteínas/genética , Sensibilidade e Especificidade , Software , Tiouridina/química , Raios UltravioletaRESUMO
Membrane fusion plays a lead role in the transport of vesicles, neurotransmission, mitochondrial dynamics, and viral infection. There are fusion proteins that catalyze and regulate the fusion. Interestingly, various types of fusion proteins are present in nature and they possess diverse mechanisms of action. We have highlighted the importance of the functional domains of intracellular heterotypic fusion, homotypic endoplasmic reticulum (ER), homotypic mitochondrial, and type-I viral fusion. During intracellular heterotypic fusion, the SNAREs and four-helix bundle formation are prevalent. Type-I viral fusion is controlled by the membrane destabilizing properties of fusion peptide and six-helix bundle formation. The ER/mitochondrial homotypic fusion is controlled by GTPase activity and the membrane destabilization properties of the amphipathic helix(s). Although the mechanism of action of these fusion proteins is diverse, they have some similarities. In all cases, the lipid composition of the membrane greatly affects membrane fusion. Next, examples of lipidation of the fusion proteins were discussed. We suggest that the fatty acyl hydrophobic tail not only acts as an anchor but may also modulate the energetics of membrane fusion intermediates. Lipidation is also important to design more effective peptide-based fusion inhibitors. Together, we have shown that membrane lipid composition and lipidation are important to modulate membrane fusion.
Assuntos
GTP Fosfo-Hidrolases , Fusão de Membrana , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , Retículo Endoplasmático/metabolismo , Dinâmica Mitocondrial , LipídeosRESUMO
The Signal Recognition Particle (SRP) and the SRP receptor (SR) are responsible for protein targeting to the plasma membrane and the protein secretory pathway. Eukaryotic SRα, one of the two proteins that form the SR, is composed of the NG, MoRF and X domains. The SRα-NG domain is responsible for binding to SRP proteins such as SRP54, interacting with RNA, binding and hydrolysing GTP. The ability to produce folded SRα-NG is a prerequisite for structural studies directed towards a better understanding of its molecular mechanism and function, as well as in (counter-)screening assays for potential binders in the drug development pipeline. However, previously reported SRα-NG constructs and purification methods only used a truncated version, lacking the first N-terminal helix. This helix in other NG domains (e.g., SRP54) has been shown to be important for protein:protein interactions but its importance in SRα remains unknown. Here, we present the cloning as well as optimised expression and purification protocols of the whole SRα-NG domain including the first N-terminal helix. We have also expressed and purified isotopically labelled SRα-NG to facilitate Nuclear Magnetic Resonance (NMR) studies.
Assuntos
GTP Fosfo-Hidrolases , Partícula de Reconhecimento de Sinal , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , Humanos , Ligação Proteica , Receptores Citoplasmáticos e Nucleares , Receptores de Peptídeos/química , Partícula de Reconhecimento de Sinal/química , Partícula de Reconhecimento de Sinal/genética , Partícula de Reconhecimento de Sinal/metabolismoRESUMO
Mitochondria are dynamic organelles that exchange contents and undergo remodelling during cyclic fusion and fission. Genetic mutations in MFN2 (the gene encoding mitofusin 2) interrupt mitochondrial fusion and cause the untreatable neurodegenerative condition Charcot-Marie-Tooth disease type 2A (CMT2A). It has not yet been possible to directly modulate mitochondrial fusion, in part because the structural basis of mitofusin function is not completely understood. Here we show that mitofusins adopt either a fusion-constrained or a fusion-permissive molecular conformation, directed by specific intramolecular binding interactions, and demonstrate that mitofusin-dependent mitochondrial fusion can be regulated in mouse cells by targeting these conformational transitions. On the basis of this model, we engineered a cell-permeant minipeptide to destabilize the fusion-constrained conformation of mitofusin and promote the fusion-permissive conformation, reversing mitochondrial abnormalities in cultured fibroblasts and neurons that harbour CMT2A-associated genetic defects. The relationship between the conformational plasticity of mitofusin 2 and mitochondrial dynamism reveals a central mechanism that regulates mitochondrial fusion, the manipulation of which can correct mitochondrial pathology triggered by defective or imbalanced mitochondrial dynamics.
Assuntos
GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/efeitos dos fármacos , Peptídeos/farmacologia , Animais , Células Cultivadas , Doença de Charcot-Marie-Tooth/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , GTP Fosfo-Hidrolases/genética , Camundongos , Mitocôndrias/genética , Mitocôndrias/patologia , Dinâmica Mitocondrial/genética , Modelos Moleculares , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Peptídeos/química , Permeabilidade , Conformação Proteica/efeitos dos fármacosRESUMO
Ribosome biogenesis is a highly complex process in eukaryotes, involving temporally and spatially regulated ribosomal protein (r-protein) binding and ribosomal RNA remodelling events in the nucleolus, nucleoplasm and cytoplasm. Hundreds of assembly factors, organized into sequential functional groups, facilitate and guide the maturation process into productive assembly branches in and across different cellular compartments. However, the precise mechanisms by which these assembly factors function are largely unknown. Here we use cryo-electron microscopy to characterize the structures of yeast nucleoplasmic pre-60S particles affinity-purified using the epitope-tagged assembly factor Nog2. Our data pinpoint the locations and determine the structures of over 20 assembly factors, which are enriched in two areas: an arc region extending from the central protuberance to the polypeptide tunnel exit, and the domain including the internal transcribed spacer 2 (ITS2) that separates 5.8S and 25S ribosomal RNAs. In particular, two regulatory GTPases, Nog2 and Nog1, act as hub proteins to interact with multiple, distant assembly factors and functional ribosomal RNA elements, manifesting their critical roles in structural remodelling checkpoints and nuclear export. Moreover, our snapshots of compositionally and structurally different pre-60S intermediates provide essential mechanistic details for three major remodelling events before nuclear export: rotation of the 5S ribonucleoprotein, construction of the active centre and ITS2 removal. The rich structural information in our structures provides a framework to dissect molecular roles of diverse assembly factors in eukaryotic ribosome assembly.