RESUMO
The type-1 ryanodine receptor (RyR1) is an intracellular calcium (Ca(2+)) release channel required for skeletal muscle contraction. Here, we present cryo-EM reconstructions of RyR1 in multiple functional states revealing the structural basis of channel gating and ligand-dependent activation. Binding sites for the channel activators Ca(2+), ATP, and caffeine were identified at interdomain interfaces of the C-terminal domain. Either ATP or Ca(2+) alone induces conformational changes in the cytoplasmic assembly ("priming"), without pore dilation. In contrast, in the presence of all three activating ligands, high-resolution reconstructions of open and closed states of RyR1 were obtained from the same sample, enabling analyses of conformational changes associated with gating. Gating involves global conformational changes in the cytosolic assembly accompanied by local changes in the transmembrane domain, which include bending of the S6 transmembrane segment and consequent pore dilation, displacement, and deformation of the S4-S5 linker and conformational changes in the pseudo-voltage-sensor domain.
Assuntos
Agonistas dos Canais de Cálcio/química , Ativação do Canal Iônico , Contração Muscular , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Animais , Sítios de Ligação , Cafeína/química , Cálcio/química , Microscopia Crioeletrônica , Ligantes , Domínios Proteicos , Coelhos , Proteínas de Ligação a Tacrolimo/químicaRESUMO
Second mitochondrial-derived activator of caspase (SMAC), also known as direct inhibitor of apoptosis-binding proteins with low pI (Diablo), is known as a pro-apoptotic mitochondrial protein released into the cytosol in response to apoptotic signals. We recently reported SMAC overexpression in cancers as essential for cell proliferation and tumor growth due to non-apoptotic functions, including phospholipid synthesis regulation. These functions may be associated with its interactions with partner proteins. Using a peptide array with 768 peptides derived from 11 selected SMAC-interacting proteins, we identified SMAC-interacting sequences. These SMAC-binding sequences were produced as cell-penetrating peptides targeted to the cytosol, mitochondria, or nucleus, inhibiting cell proliferation and inducing apoptosis in several cell lines. For in vivo study, a survivin/baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5)-derived peptide was selected, due to its overexpression in many cancers and its involvement in mitosis, apoptosis, autophagy, cell proliferation, inflammation, and immune responses, as a target for cancer therapy. Specifically, a SMAC-targeting survivin/BIRC5-derived peptide, given intratumorally or intravenously, strongly inhibited lung tumor growth, cell proliferation, angiogenesis, and inflammation, induced apoptosis, and remodeled the tumor microenvironment. The peptide promoted tumor infiltration of CD-8+ cells and increased cell-intrinsic programmed cell death protein 1 (PD-1) and programmed cell death ligand 1 (PD-L1) expression, resulting in cancer cell self-destruction and increased tumor cell death, preserving immune cells. Thus, targeting the interaction between the multifunctional proteins SMAC and survivin represents an innovative therapeutic cancer paradigm.
Assuntos
Proteínas Reguladoras de Apoptose , Apoptose , Proliferação de Células , Proteínas Mitocondriais , Survivina , Humanos , Survivina/metabolismo , Survivina/genética , Animais , Camundongos , Proteínas Mitocondriais/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Reguladoras de Apoptose/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias/metabolismo , Neoplasias/patologia , Neoplasias/tratamento farmacológico , Inflamação/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Ligação Proteica , Proteínas Inibidoras de Apoptose/metabolismo , Proteínas Inibidoras de Apoptose/genética , Peptídeos Penetradores de Células/farmacologia , Peptídeos Penetradores de Células/química , Peptídeos/farmacologia , Peptídeos/química , Terapia de ImunossupressãoRESUMO
In recent years, there has been growing interest in SARM1 as a potential breakthrough drug target for treating various pathologies of axon degeneration. SARM1-mediated axon degeneration relies on its TIR domain NADase activity, but recent structural data suggest that the non-catalytic ARM domain could also serve as a pharmacological site as it has an allosteric inhibitory function. Here, we screened for synthetic small molecules that inhibit SARM1, and tested a selected set of these compounds in a DRG axon degeneration assay. Using cryo-EM, we found that one of the newly discovered inhibitors, a calmidazolium designated TK106, not only stabilizes the previously reported inhibited conformation of the octamer, but also a meta-stable structure: a duplex of octamers (16 protomers), which we have now determined to 4.0 Å resolution. In the duplex, each ARM domain protomer is engaged in lateral interactions with neighboring protomers, and is further stabilized by contralateral contacts with the opposing octamer ring. Mutagenesis of the duplex contact sites leads to a moderate increase in SARM1 activation in cultured cells. Based on our data we propose that the duplex assembly constitutes an additional auto-inhibition mechanism that tightly prevents pre-mature activation and axon degeneration.
Assuntos
Proteínas do Domínio Armadillo , Axônios , Axônios/metabolismo , Subunidades Proteicas , Células Cultivadas , Domínios Proteicos , Proteínas do Domínio Armadillo/metabolismo , MutagêneseRESUMO
High-resolution structures are crucial for understanding the functional properties of nanomaterials. We applied single-particle cryo-electron microscopy (cryo-EM), a method traditionally used for structure determination of biological macromolecules, to obtain high-resolution structures of synthetic non-biological filaments formed by photopolymerization of macrocyclic diacetylene (MDA) amphiphilic monomers. Tomographic analysis showed that the MDA monomers self-assemble into hollow nanotubes upon dispersion in water. Single-particle analysis revealed tubes consisting of six pairs of covalently bonded filaments held together by hydrophobic interactions, where each filament is composed of macrocyclic rings stacked in parallel "chair" conformations. The hollow MDA nanotube structures we found may account for the efficient scavenging of amphiphilic pollutants in water and subsequent photodegradation of the guest species.
Assuntos
Poluentes Ambientais , Nanotubos , Microscopia Crioeletrônica/métodos , Polímero Poliacetilênico , ÁguaRESUMO
Impaired mitochondrial function has been proposed as a causative factor in neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), caused by motor neuron degeneration. Mutations in superoxide dismutase (SOD1) cause ALS and SOD1 mutants were shown to interact with the voltage-dependent anion channel 1 (VDAC1), affecting its normal function. VDAC1 is a multi-functional channel located at the outer mitochondrial membrane that serves as a mitochondrial gatekeeper controlling metabolic and energetic crosstalk between mitochondria and the rest of the cell and it is a key player in mitochondria-mediated apoptosis. Previously, we showed that VDAC1 interacts with SOD1 and that the VDAC1-N-terminal-derived peptide prevented mutant SOD1 cytotoxic effects. In this study, using a peptide array, we identified the SOD1 sequence that interacts with VDAC1. Synthetic peptides generated from the identified VDAC1-binding sequences in SOD1 directly interacted with purified VDAC1. We also show that VDAC1 oligomerization increased in spinal cord mitochondria isolated from mutant SOD1G93A mice and rats. Thus, we used the novel VDAC1-specific small molecules, VBIT-4 and VBIT-12, inhibiting VDAC1 oligomerization and subsequently apoptosis and associated processes such as ROS production, and increased cytosolic Ca2+. VBIT-12 was able to rescue cell death induced by mutant SOD1 in neuronal cultures. Finally, although survival was not affected, VBIT-12 administration significantly improved muscle endurance in mutant SOD1G93A mice. Therefore, VBIT-12 may represent an attractive therapy for maintaining muscle function during the progression of ALS.
Assuntos
Esclerose Lateral Amiotrófica , Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Transgênicos , Proteínas Mitocondriais/metabolismo , Ratos , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Canal de Ânion 1 Dependente de Voltagem/genética , Canal de Ânion 1 Dependente de Voltagem/metabolismoRESUMO
Ryanodine receptors (RyRs) are calcium release channels expressed in the sarcoendoplasmic reticula of many cell types including cardiac and skeletal muscle cells. In recent years Ca2+ leak through RyRs has been implicated as a major contributor to the development of diseases including heart failure, muscle myopathies, Alzheimer's disease, and diabetes, making it an important therapeutic target. Recent mammalian RyR1 cryoelectron microscopy (cryo-EM) structures of multiple functional states have clarified longstanding questions including the architecture of the transmembrane (TM) pore and cytoplasmic domains, the location and architecture of the channel gate, ligand-binding sites, and the gating mechanism. As we advance toward complete models of RyRs this new information enables the determination of domain-domain interfaces and the location and structural effects of disease-causing RyR mutations.
Assuntos
Cálcio/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Animais , Humanos , Modelos Moleculares , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Canal de Liberação de Cálcio do Receptor de Rianodina/genéticaRESUMO
Ryanodine receptors (RyRs) mediate the rapid release of calcium (Ca(2+)) from intracellular stores into the cytosol, which is essential for numerous cellular functions including excitation-contraction coupling in muscle. Lack of sufficient structural detail has impeded understanding of RyR gating and regulation. Here we report the closed-state structure of the 2.3-megadalton complex of the rabbit skeletal muscle type 1 RyR (RyR1), solved by single-particle electron cryomicroscopy at an overall resolution of 4.8 Å. We fitted a polyalanine-level model to all 3,757 ordered residues in each protomer, defining the transmembrane pore in unprecedented detail and placing all cytosolic domains as tertiary folds. The cytosolic assembly is built on an extended α-solenoid scaffold connecting key regulatory domains to the pore. The RyR1 pore architecture places it in the six-transmembrane ion channel superfamily. A unique domain inserted between the second and third transmembrane helices interacts intimately with paired EF-hands originating from the α-solenoid scaffold, suggesting a mechanism for channel gating by Ca(2+).
Assuntos
Canal de Liberação de Cálcio do Receptor de Rianodina/química , Canal de Liberação de Cálcio do Receptor de Rianodina/ultraestrutura , Animais , Cálcio/deficiência , Cálcio/metabolismo , Cálcio/farmacologia , Membrana Celular/metabolismo , Microscopia Crioeletrônica , Citosol/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Músculo Esquelético/química , Estrutura Terciária de Proteína , Coelhos , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Proteínas de Ligação a Tacrolimo/química , Proteínas de Ligação a Tacrolimo/metabolismoRESUMO
Single-channel recordings are widely used to explore functional properties of ion channels. Typically, such recordings are performed at bandwidths of less than 10 kHz because of signal-to-noise considerations, limiting the temporal resolution available for studying fast gating dynamics to greater than 100 µs. Here we present experimental methods that directly integrate suspended lipid bilayers with high-bandwidth, low-noise transimpedance amplifiers based on complementary metal-oxide-semiconductor (CMOS) integrated circuits (IC) technology to achieve bandwidths in excess of 500 kHz and microsecond temporal resolution. We use this CMOS-integrated bilayer system to study the type 1 ryanodine receptor (RyR1), a Ca2+-activated intracellular Ca2+-release channel located on the sarcoplasmic reticulum. We are able to distinguish multiple closed states not evident with lower bandwidth recordings, suggesting the presence of an additional Ca2+ binding site, distinct from the site responsible for activation. An extended beta distribution analysis of our high-bandwidth data can be used to infer closed state flicker events as fast as 35 ns. These events are in the range of single-file ion translocations.
Assuntos
Bicamadas Lipídicas/química , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Semicondutores , Sinalização do Cálcio , Membrana Celular , Técnicas Eletroquímicas , Ativação do Canal Iônico , Metais/química , Óxidos/química , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Fatores de TempoRESUMO
Biomineralization is mediated by specialized proteins that guide and control mineral sedimentation. In many cases, the active regions of these biomineralization proteins are intrinsically disordered. High-resolution structures of these proteins while they interact with minerals are essential for understanding biomineralization processes and the function of intrinsically disordered proteins (IDPs). Here we used the cavity of ferritin as a nanoreactor where the interaction between M6A, an intrinsically disordered iron-binding domain, and an iron oxide particle was visualized at high resolution by cryo-EM. Taking advantage of the differences in the electron-dose sensitivity of the protein and the iron oxide particles, we developed a method to determine the irregular shape of the particles found in our density maps. We found that the folding of M6A correlates with the detection of mineral particles in its vicinity. M6A interacts with the iron oxide particles through its C-terminal side, resulting in the stabilization of a helix at its N-terminal side. The stabilization of the helix at a region that is not in direct contact with the iron oxide particle demonstrates the ability of IDPs to respond to signals from their surroundings by conformational changes. These findings provide the first glimpse toward the long-suspected mechanism for biomineralization protein control over mineral microstructure, where unstructured regions of these proteins become more ordered in response to their interaction with the nascent mineral particles.
Assuntos
Apoferritinas/química , Proteínas de Bactérias/química , Microscopia Crioeletrônica/métodos , Compostos Férricos/química , Proteínas Intrinsicamente Desordenadas/química , Peptídeos/química , Sítios de Ligação , Biomineralização , Nanopartículas Magnéticas de Óxido de Ferro/química , Magnetospirillum/química , Modelos Moleculares , Tamanho da Partícula , Ligação Proteica , Conformação Proteica , Dobramento de ProteínaRESUMO
Alteration of ryanodine receptor (RyR)-mediated calcium (Ca2+) signaling has been reported in Alzheimer disease (AD) models. However, the molecular mechanisms underlying altered RyR-mediated intracellular Ca2+ release in AD remain to be fully elucidated. We report here that RyR2 undergoes post-translational modifications (phosphorylation, oxidation, and nitrosylation) in SH-SY5Y neuroblastoma cells expressing the ß-amyloid precursor protein (ßAPP) harboring the familial double Swedish mutations (APPswe). RyR2 macromolecular complex remodeling, characterized by depletion of the regulatory protein calstabin2, resulted in increased cytosolic Ca2+ levels and mitochondrial oxidative stress. We also report a functional interplay between amyloid ß (Aß), ß-adrenergic signaling, and altered Ca2+ signaling via leaky RyR2 channels. Thus, post-translational modifications of RyR occur downstream of Aß through a ß2-adrenergic signaling cascade that activates PKA. RyR2 remodeling in turn enhances ßAPP processing. Importantly, pharmacological stabilization of the binding of calstabin2 to RyR2 channels, which prevents Ca2+ leakage, or blocking the ß2-adrenergic signaling cascade reduced ßAPP processing and the production of Aß in APPswe-expressing SH-SY5Y cells. We conclude that targeting RyR-mediated Ca2+ leakage may be a therapeutic approach to treat AD.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Sinalização do Cálcio , Neurônios/enzimologia , Processamento de Proteína Pós-Traducional , Receptores Adrenérgicos beta 2/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Antagonistas de Receptores Adrenérgicos beta 2/farmacologia , Doença de Alzheimer/enzimologia , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/genética , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular Tumoral , Proteínas Quinases Dependentes de AMP Cíclico/química , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Humanos , Mutação , Proteínas do Tecido Nervoso/agonistas , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Proteínas de Ligação a Tacrolimo/antagonistas & inibidores , Proteínas de Ligação a Tacrolimo/metabolismoRESUMO
The mechanisms underlying ryanodine receptor (RyR) dysfunction associated with Alzheimer disease (AD) are still not well understood. Here, we show that neuronal RyR2 channels undergo post-translational remodeling (PKA phosphorylation, oxidation, and nitrosylation) in brains of AD patients, and in two murine models of AD (3 × Tg-AD, APP +/- /PS1 +/-). RyR2 is depleted of calstabin2 (KFBP12.6) in the channel complex, resulting in endoplasmic reticular (ER) calcium (Ca2+) leak. RyR-mediated ER Ca2+ leak activates Ca2+-dependent signaling pathways, contributing to AD pathogenesis. Pharmacological (using a novel RyR stabilizing drug Rycal) or genetic rescue of the RyR2-mediated intracellular Ca2+ leak improved synaptic plasticity, normalized behavioral and cognitive functions and reduced Aß load. Genetically altered mice with congenitally leaky RyR2 exhibited premature and severe defects in synaptic plasticity, behavior and cognitive function. These data provide a mechanism underlying leaky RyR2 channels, which could be considered as potential AD therapeutic targets.
Assuntos
Doença de Alzheimer/metabolismo , Cálcio/metabolismo , Transtornos Cognitivos/metabolismo , Processamento de Proteína Pós-Traducional , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Doença de Alzheimer/patologia , Animais , Sinalização do Cálcio , Transtornos Cognitivos/patologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Feminino , Humanos , Masculino , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Transgênicos , Estresse Oxidativo/fisiologia , Fosforilação , Reconhecimento Psicológico/fisiologia , Retículo Sarcoplasmático/metabolismoRESUMO
The swimming device of archaea-the archaellum-presents asparagine (N)-linked glycans. While N-glycosylation serves numerous roles in archaea, including enabling their survival in extreme environments, how this post-translational modification contributes to cell motility remains under-explored. Here, we report the cryo-EM structure of archaellum filaments from the haloarchaeon Halobacterium salinarum, where archaellins, the building blocks of the archaellum, are N-glycosylated, and the N-glycosylation pathway is well-resolved. We further determined structures of archaellum filaments from two N-glycosylation mutant strains that generate truncated glycans and analyzed their motility. While cells from the parent strain exhibited unidirectional motility, the N-glycosylation mutant strain cells swam in ever-changing directions within a limited area. Although these mutant strain cells presented archaellum filaments that were highly similar in architecture to those of the parent strain, N-linked glycan truncation greatly affected interactions between archaellum filaments, leading to dramatic clustering of both isolated and cell-attached filaments. We propose that the N-linked tetrasaccharides decorating archaellins act as physical spacers that minimize the archaellum filament aggregation that limits cell motility.
Assuntos
Proteínas Arqueais , Halobacterium salinarum , Glicosilação , Halobacterium salinarum/metabolismo , Halobacterium salinarum/genética , Proteínas Arqueais/metabolismo , Proteínas Arqueais/genética , Proteínas Arqueais/química , Polissacarídeos/metabolismo , Microscopia Crioeletrônica , Mutação , Citoesqueleto/metabolismo , Processamento de Proteína Pós-Traducional , Movimento CelularRESUMO
Several kinesin-5 motors (kinesin-5s) exhibit bidirectional motility. The mechanism of such motility remains unknown. Bidirectional kinesin-5s share a long N-terminal nonmotor domain (NTnmd), absent in exclusively plus-end-directed kinesins. Here, we combined in vivo, in vitro, and cryo-electron microscopy (cryo-EM) studies to examine the impact of NTnmd mutations on the motor functions of the bidirectional kinesin-5, Cin8. We found that NTnmd deletion mutants exhibited cell viability and spindle localization defects. Using cryo-EM, we examined the structure of a microtubule (MT)-bound motor domain of Cin8, containing part of its NTnmd. Modeling and molecular dynamic simulations based on the cryo-EM map suggested that the NTnmd of Cin8 interacts with the C-terminal tail of ß-tubulin. In vitro experiments on subtilisin-treated MTs confirmed this notion. Last, we showed that NTnmd mutants are defective in plus-end-directed motility in single-molecule and antiparallel MT sliding assays. These findings demonstrate that the NTnmd, common to bidirectional kinesin-5s, is critical for their bidirectional motility and intracellular functions.
Assuntos
Cinesinas , Proteínas de Saccharomyces cerevisiae , Cinesinas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Microscopia Crioeletrônica , Microtúbulos/químicaRESUMO
The interaction between nuclear receptor coactivator 4 (NCOA4) and the iron storage protein ferritin is a crucial component of cellular iron homeostasis. The binding of NCOA4 to the FTH1 subunits of ferritin initiates ferritinophagy-a ferritin-specific autophagic pathway leading to the release of the iron stored inside ferritin. The dysregulation of NCOA4 is associated with several diseases, including neurodegenerative disorders and cancer, highlighting the NCOA4-ferritin interface as a prime target for drug development. Here, we present the cryo-EM structure of the NCOA4-FTH1 interface, resolving 16 amino acids of NCOA4 that are crucial for the interaction. The characterization of mutants, designed to modulate the NCOA4-FTH1 interaction, is used to validate the significance of the different features of the binding site. Our results explain the role of the large solvent-exposed hydrophobic patch found on the surface of FTH1 and pave the way for the rational development of ferritinophagy modulators.
Assuntos
Microscopia Crioeletrônica , Ferritinas , Coativadores de Receptor Nuclear , Ferritinas/metabolismo , Ferritinas/química , Ferritinas/genética , Humanos , Coativadores de Receptor Nuclear/metabolismo , Coativadores de Receptor Nuclear/química , Coativadores de Receptor Nuclear/genética , Ligação Proteica , Sítios de Ligação , Ferro/metabolismo , Autofagia , Modelos Moleculares , Células HEK293 , Oxirredutases/metabolismo , Oxirredutases/química , Oxirredutases/genética , Proteólise , MutaçãoRESUMO
Microbial taxonomy is critical for describing ecosystem composition, yet the link between taxonomy and properties of microbes, such as their cellular architecture, remains poorly defined. We hypothesized that the cellular architecture represents microbial niche adaptation. We used cryo-electron microscopy and tomography to analyze microbial morphology in order to associate cellular architecture with phylogeny and genomic contents. As a model system, we chose the core rumen microbiome and imaged a large isolate collection covering 90% of its richness at the order level. Based on quantifications of several morphological features, we found that the visual similarity of microbiota is significantly related to their phylogenetic distance. Up to the Family level, closely related microbes have similar cellular architectures, which are highly correlated with genome similarity. However, in more distantly related bacteria, the correlation both with taxonomy and genome similarity is lost. This is the first comprehensive study of microbial cellular architecture and our results highlight that structure remains an important parameter in classification of microorganisms, along with functional parameters such as metabolomics. Furthermore, the high-quality images presented in this study represent a reference database for the identification of bacteria in anaerobic ecosystems.
RESUMO
Wallerian axonal degeneration (WD) does not occur in the nematode C. elegans, in contrast to other model animals. However, WD depends on the NADase activity of SARM1, a protein that is also expressed in C. elegans (ceSARM/ceTIR-1). We hypothesized that differences in SARM between species might exist and account for the divergence in WD. We first show that expression of the human (h)SARM1, but not ceTIR-1, in C. elegans neurons is sufficient to confer axon degeneration after nerve injury. Next, we determined the cryoelectron microscopy structure of ceTIR-1 and found that, unlike hSARM1, which exists as an auto-inhibited ring octamer, ceTIR-1 forms a readily active 9-mer. Enzymatically, the NADase activity of ceTIR-1 is substantially weaker (10-fold higher Km) than that of hSARM1, and even when fully active, it falls short of consuming all cellular NAD+. Our experiments provide insight into the molecular mechanisms and evolution of SARM orthologs and WD across species.
Assuntos
Axônios , Caenorhabditis elegans , Animais , Humanos , Axônios/metabolismo , Caenorhabditis elegans/metabolismo , Microscopia Crioeletrônica , Neurônios/metabolismo , Proteínas do Domínio Armadillo/metabolismo , NAD+ Nucleosidase/metabolismo , Degeneração Walleriana/metabolismoRESUMO
We report a new type of nanoparticle, consisting of a nucleic acid core (>7500 nt) folded into a 35 nm DNA origami sphere, encapsulated by a capsid composed of all three SV40 virus capsid proteins. Compared to the prototype reported previously, whose capsid consists of VP1 only, the new nanoparticle closely adopts the unique intracellular pathway of the native SV40, suggesting that the proteins of the synthetic capsid retain their native viral functionality. Some of the challenges in the design of such near-future composite drugs destined for gene delivery are discussed.
Assuntos
Capsídeo , Vírus , Proteínas do Capsídeo/metabolismo , DNA/metabolismo , DNA Viral/metabolismo , Vírus 40 dos Símios , Vírion , Montagem de Vírus , Vírus/metabolismoRESUMO
SMAC/Diablo, a pro-apoptotic protein, yet it is overexpressed in several cancer types. We have described a noncanonical function for SMAC/Diablo as a regulator of lipid synthesis during cancer cell proliferation and development. Here, we explore the molecular mechanism through which SMAC/Diablo regulates phospholipid synthesis. We showed that SMAC/Diablo directly interacts with mitochondrial phosphatidylserine decarboxylase (PSD) and inhibits its catalytic activity during synthesis of phosphatidylethanolamine (PE) from phosphatidylserine (PS). Unlike other phospholipids (PLs), PE is synthesized not only in the endoplasmic reticulum but also in mitochondria. As a result, PSD activity and mitochondrial PE levels were increased in the mitochondria of SMAC/Diablo-deficient cancer cells, with the total amount of cellular PLs and phosphatidylcholine (PC) being lower as compared to SMAC-expressing cancer cells. Moreover, in the absence of SMAC/Diablo, PSD inhibited cancer cell proliferation by catalysing the overproduction of mitochondrial PE and depleting the cellular levels of PC, PE and PS. Additionally, we demonstrated that both SMAC/Diablo and PSD colocalization in the nucleus resulted in increased levels of nuclear PE, that acts as a signalling molecule in regulating several nuclear activities. By using a peptide array composed of 768-peptides derived from 11 SMAC-interacting proteins, we identified six nuclear proteins ARNT, BIRC2, MAML2, NR4A1, BIRC5 and HTRA2 Five of them also interacted with PSD through motifs that are not involved in SMAC binding. Synthetic peptides carrying the PSD-interacting motifs of these proteins could bind purified PSD and inhibit the PSD catalytic activity. When targeted specifically to the mitochondria or the nucleus, these synthetic peptides inhibited cancer cell proliferation. To our knowledge, these are the first reported inhibitors of PSD acting also as inhibitors of cancer cell proliferation. Altogether, we demonstrated that phospholipid metabolism and PE synthesis regulated by the SMAC-PSD interaction are essential for cancer cell proliferation and may be potentially targeted for treating cancer.