RESUMO
Natural photosystems couple light harvesting to charge separation using a 'special pair' of chlorophyll molecules that accepts excitation energy from the antenna and initiates an electron-transfer cascade. To investigate the photophysics of special pairs independently of the complexities of native photosynthetic proteins, and as a first step toward creating synthetic photosystems for new energy conversion technologies, we designed C2-symmetric proteins that hold two chlorophyll molecules in closely juxtaposed arrangements. X-ray crystallography confirmed that one designed protein binds two chlorophylls in the same orientation as native special pairs, whereas a second designed protein positions them in a previously unseen geometry. Spectroscopy revealed that the chlorophylls are excitonically coupled, and fluorescence lifetime imaging demonstrated energy transfer. The cryo-electron microscopy structure of a designed 24-chlorophyll octahedral nanocage with a special pair on each edge closely matched the design model. The results suggest that the de novo design of artificial photosynthetic systems is within reach of current computational methods.
Assuntos
Clorofila , Clorofila/química , Clorofila/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Fotossíntese , Transferência de Energia , Microscopia Crioeletrônica , Conformação Proteica , Complexos de Proteínas Captadores de Luz/química , Complexos de Proteínas Captadores de Luz/metabolismoRESUMO
Mitochondrial derangement is an important contributor to the pathophysiology of muscular dystrophies and may be among the earliest cellular deficits. We have previously shown that disruption of Mss51, a mammalian skeletal muscle protein that localizes to the mitochondria, results in enhanced muscle oxygen consumption rate, increased endurance capacity, and improved limb muscle strength in mice with wildtype background. Here, we investigate whether Mss51 deletion in the mdx murine model of Duchenne muscular dystrophy (mdx-Mss51 KO) counteracts the muscle pathology and mitochondrial irregularities observed in mdx mice. We found that mdx-Mss51 KO mice had increased myofiber oxygen consumption rates and an amelioration of muscle histopathology compared to mdx counterparts. This corresponded with greater treadmill endurance and less percent fatigue in muscle physiology, but no improvement in forelimb grip strength or limb muscle force production. These findings suggest that although Mss51 deletion ameliorates the skeletal muscle mitochondrial respiration defects in mdx and improves fatigue resistance in vivo, the lack of improvement in force production suggests that this target alone may be insufficient for a therapeutic effect.
Assuntos
Deleção de Genes , Proteínas Mitocondriais/genética , Força Muscular , Distrofia Muscular de Duchenne/genética , Fatores de Transcrição/genética , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Mitocôndrias Musculares/metabolismo , Mitocôndrias Musculares/patologia , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologia , Consumo de OxigênioRESUMO
Peptides constrained by intramolecular cross-links, especially stapled α-helices, have emerged as versatile scaffolds for drug development. However, there are fewer examples of similarly constrained scaffolds for other secondary structures. Here, we used a novel computational strategy to identify an optimal staple for antiparallel ß-strands, and then we incorporated that staple within a ß-hairpin peptide. The hairpin uses 4-mercaptoproline as a novel staple component, which contributes to a unique, kinked structure. The stapled hairpins show a high degree of structure in aqueous solution, excellent resistance to degradation in cell lysates, and cytosolic penetration at micromolar concentrations. They also overlay with a unique subset of kinked hairpin motifs at protein-protein interaction interfaces. Thus, these scaffolds represent promising starting points for developing inhibitors of cellular protein-protein interactions.
Assuntos
Peptídeos/síntese química , Prolina/análogos & derivados , Sequência de Aminoácidos , Modelos Moleculares , Peptídeos/química , Prolina/química , Estrutura Secundária de ProteínaRESUMO
Small macrocycles with four or fewer amino acids are among the most potent natural products known, but there is currently no way to systematically generate such compounds. We describe a computational method for identifying ordered macrocycles composed of alpha, beta, gamma, and 17 other amino acid backbone chemistries, which we used to predict 14.9 million closed cycles composed of >42,000 monomer combinations. We chemically synthesized 18 macrocycles predicted to adopt single low-energy states and determined their x-ray or nuclear magnetic resonance structures; 15 of these were very close to the design models. We illustrate the therapeutic potential of these macrocycle designs by developing selective inhibitors of three protein targets of current interest. By opening up a vast space of readily synthesizable drug-like macrocycles, our results should considerably enhance structure-based drug design.
Assuntos
Amidas , Aminoácidos , Produtos Biológicos , Desenho de Fármacos , Peptídeos Cíclicos , Amidas/química , Aminoácidos/química , Produtos Biológicos/síntese química , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Cristalografia por Raios X , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Molecular , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologiaRESUMO
Natural photosystems couple light harvesting to charge separation using a "special pair" of chlorophyll molecules that accepts excitation energy from the antenna and initiates an electron-transfer cascade. To investigate the photophysics of special pairs independent of complexities of native photosynthetic proteins, and as a first step towards synthetic photosystems for new energy conversion technologies, we designed C2-symmetric proteins that precisely position chlorophyll dimers. X-ray crystallography shows that one designed protein binds two chlorophylls in a binding orientation matching native special pairs, while a second positions them in a previously unseen geometry. Spectroscopy reveals excitonic coupling, and fluorescence lifetime imaging demonstrates energy transfer. We designed special pair proteins to assemble into 24-chlorophyll octahedral nanocages; the design model and cryo-EM structure are nearly identical. The design accuracy and energy transfer function of these special pair proteins suggest that de novo design of artificial photosynthetic systems is within reach of current computational methods.
RESUMO
A method for obtaining the intensity fluctuation spectra of dynamic laser speckle patterns is introduced, which is based on the temporal modulation of the illumination and the subsequent integration of the intensity signals. This approach does not rely on the fast sampling rate to meet the Nyquist criterion, making it applicable for full-field imaging applications. The intensity fluctuation spectra created by the in-plane motion of a random phase object was investigated by using both a single-channel detector and a multichannel sensor. The power spectra obtained by using the full-field temporal modulation method were found to agree with the homodyne Doppler spectra obtained by using the method of autocorrelation and Fourier transform.
RESUMO
Peptide heterodimers are prevalent in nature, which are not only functional macromolecules but molecular tools for chemical and synthetic biology. Computational methods have also been developed to design heterodimers of advanced functions. However, these peptide heterodimers are usually formed through noncovalent interactions, which are prone to dissociate and subject to concentration-dependent nonspecific aggregation. Heterodimers crosslinked with interchain disulfide bonds are more stable, but it represents a formidable challenge for both the computational design of heterodimers and the manipulation of disulfide pairing for heterodimer synthesis and applications. Here, we report the design, synthesis and application of interchain disulfide-bridged peptide heterodimers with mutual orthogonality by combining computational de novo designs with a directed disulfide pairing strategy. These heterodimers can be used as not only scaffolds for generating functional molecules but chemical tools or building blocks for protein labeling and construction of crosslinking hybrids. This study thus opens the door for using this unexplored dimeric structure space for many biological applications.
Assuntos
Dissulfetos , Peptídeos , Dissulfetos/química , Substâncias Macromoleculares , Peptídeos/química , Dobramento de Proteína , ProteínasRESUMO
Antibodies, and antibody derivatives such as nanobodies, contain immunoglobulin-like (Ig) ß-sandwich scaffolds which anchor the hypervariable antigen-binding loops and constitute the largest growing class of drugs. Current engineering strategies for this class of compounds rely on naturally existing Ig frameworks, which can be hard to modify and have limitations in manufacturability, designability and range of action. Here, we develop design rules for the central feature of the Ig fold architecture-the non-local cross-ß structure connecting the two ß-sheets-and use these to design highly stable Ig domains de novo, confirm their structures through X-ray crystallography, and show they can correctly scaffold functional loops. Our approach opens the door to the design of antibody-like scaffolds with tailored structures and superior biophysical properties.
Assuntos
Anticorpos de Domínio Único , Sequência de Aminoácidos , Anticorpos/química , Regiões Determinantes de Complementaridade , Domínios de Imunoglobulina , Modelos Moleculares , Conformação ProteicaRESUMO
PURPOSE OF REVIEW: This review evaluates recently published literature examining various muscle tissue cells and their modulators that determine whether injured skeletal muscle will fully regenerate or become fibrotic. RECENT FINDINGS: Muscle regeneration is a complex process involving several interacting cell types. Macrophages initiate a cytokine response to injury that both directs the subsequent inflammatory response and promotes nonmyeloid proliferation. Muscle cells and their progenitors produce autocrine and paracrine growth factors that help inhibit or stimulate muscle growth and regeneration. Cells of the connective tissue, including fibroblasts and newly described fibro/adipogenic progenitors, can support myogenic cells and remodel the extracellular matrix. However in certain environments, fibrosis can become a self-perpetuating process leading to incomplete muscle regeneration. SUMMARY: Several cell types are involved in the muscle repair process, interacting through multiple signaling molecules and pathways. This provides a richness of potential therapeutic targets to reduce fibrosis and facilitate skeletal muscle regeneration.
Assuntos
Músculo Esquelético/patologia , Músculo Esquelético/fisiologia , Regeneração/fisiologia , Animais , Células do Tecido Conjuntivo/patologia , Células do Tecido Conjuntivo/fisiologia , Fibrose , Humanos , Fibras Musculares Esqueléticas/patologia , Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/imunologia , Músculo Esquelético/lesões , Mioblastos Esqueléticos/patologia , Mioblastos Esqueléticos/fisiologia , Regeneração/imunologia , Transdução de SinaisRESUMO
Despite recent success in computational design of structured cyclic peptides, de novo design of cyclic peptides that bind to any protein functional site remains difficult. To address this challenge, we develop a computational "anchor extension" methodology for targeting protein interfaces by extending a peptide chain around a non-canonical amino acid residue anchor. To test our approach using a well characterized model system, we design cyclic peptides that inhibit histone deacetylases 2 and 6 (HDAC2 and HDAC6) with enhanced potency compared to the original anchor (IC50 values of 9.1 and 4.4 nM for the best binders compared to 5.4 and 0.6 µM for the anchor, respectively). The HDAC6 inhibitor is among the most potent reported so far. These results highlight the potential for de novo design of high-affinity protein-peptide interfaces, as well as the challenges that remain.
Assuntos
Desenho de Fármacos , Inibidores de Histona Desacetilases/farmacologia , Peptídeos Cíclicos/farmacologia , Relação Estrutura-Atividade , Domínio Catalítico/efeitos dos fármacos , Cristalografia por Raios X , Ensaios Enzimáticos , Histona Desacetilase 2/antagonistas & inibidores , Histona Desacetilase 2/isolamento & purificação , Histona Desacetilase 2/metabolismo , Histona Desacetilase 2/ultraestrutura , Desacetilase 6 de Histona/antagonistas & inibidores , Desacetilase 6 de Histona/genética , Desacetilase 6 de Histona/isolamento & purificação , Desacetilase 6 de Histona/ultraestrutura , Inibidores de Histona Desacetilases/química , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , Ressonância Magnética Nuclear Biomolecular , Biblioteca de Peptídeos , Peptídeos Cíclicos/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/ultraestruturaRESUMO
Multivalent presentation of viral glycoproteins can substantially increase the elicitation of antigen-specific antibodies. To enable a new generation of anti-viral vaccines, we designed self-assembling protein nanoparticles with geometries tailored to present the ectodomains of influenza, HIV, and RSV viral glycoprotein trimers. We first de novo designed trimers tailored for antigen fusion, featuring N-terminal helices positioned to match the C termini of the viral glycoproteins. Trimers that experimentally adopted their designed configurations were incorporated as components of tetrahedral, octahedral, and icosahedral nanoparticles, which were characterized by cryo-electron microscopy and assessed for their ability to present viral glycoproteins. Electron microscopy and antibody binding experiments demonstrated that the designed nanoparticles presented antigenically intact prefusion HIV-1 Env, influenza hemagglutinin, and RSV F trimers in the predicted geometries. This work demonstrates that antigen-displaying protein nanoparticles can be designed from scratch, and provides a systematic way to investigate the influence of antigen presentation geometry on the immune response to vaccination.
Vaccines train the immune system to recognize a specific virus or bacterium so that the body can be better prepared against these harmful agents. To do so, many vaccines contain viral molecules called glycoproteins, which are specific to each type of virus. Glycoproteins that sit at the surface of the virus can act as 'keys' that recognize and unlock the cells of certain organisms, leading to viral infection. To ensure a stronger immune response, glycoproteins in vaccines are often arranged on a protein scaffolding which can mimic the shape of the virus of interest and trigger a strong immune response. Many scaffoldings, however, are currently made from natural proteins which cannot always display viral glycoproteins. Here, Ueda, Antanasijevic et al. developed a method that allows for the design of artificial proteins which can serve as scaffolding for viral glycoproteins. This approach was tested using three viruses: influenza, HIV, and RSV a virus responsible for bronchiolitis. The experiments showed that in each case, the relevant viral glycoproteins could attach themselves to the scaffolding. These structures could then assemble themselves into vaccine particles with predicted geometrical shapes, which mimicked the virus and maximized the response from the immune system. Designing artificial scaffolding for viral glycoproteins gives greater control over vaccine design, allowing scientists to manipulate the shape of vaccine particles and test the impact on the immune response. Ultimately, the approach developed by Ueda, Antanasijevic et al. could lead to vaccines that are more efficient and protective, including against viruses for which there is currently no suitable scaffolding.
Assuntos
Antígenos Virais/imunologia , Glicoproteínas/imunologia , Imunidade Humoral , Vacinas contra Influenza/imunologia , Nanopartículas/química , Antígenos Virais/química , Microscopia Crioeletrônica , Glicoproteínas/química , Humanos , Vacinas contra Influenza/química , VacinaçãoRESUMO
Myostatin is a negative regulator of muscle growth and metabolism and its inhibition in mice improves insulin sensitivity, increases glucose uptake into skeletal muscle, and decreases total body fat. A recently described mammalian protein called MSS51 is significantly downregulated with myostatin inhibition. In vitro disruption of Mss51 results in increased levels of ATP, ß-oxidation, glycolysis, and oxidative phosphorylation. To determine the in vivo biological function of Mss51 in mice, we disrupted the Mss51 gene by CRISPR/Cas9 and found that Mss51-KO mice have normal muscle weights and fiber-type distribution but reduced fat pads. Myofibers isolated from Mss51-KO mice showed an increased oxygen consumption rate compared with WT controls, indicating an accelerated rate of skeletal muscle metabolism. The expression of genes related to oxidative phosphorylation and fatty acid ß-oxidation were enhanced in skeletal muscle of Mss51-KO mice compared with that of WT mice. We found that mice lacking Mss51 and challenged with a high-fat diet were resistant to diet-induced weight gain, had increased whole-body glucose turnover and glycolysis rate, and increased systemic insulin sensitivity and fatty acid ß-oxidation. These findings demonstrate that MSS51 modulates skeletal muscle mitochondrial respiration and regulates whole-body glucose and fatty acid metabolism, making it a potential target for obesity and diabetes.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Glucose/metabolismo , Proteínas Mitocondriais/deficiência , Fibras Musculares Esqueléticas/metabolismo , Obesidade/metabolismo , Fatores de Transcrição/deficiência , Animais , Sistemas CRISPR-Cas/genética , Diabetes Mellitus Tipo 2/etiologia , Diabetes Mellitus Tipo 2/genética , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Ácidos Graxos/metabolismo , Feminino , Humanos , Insulina , Resistência à Insulina/genética , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Fibras Musculares Esqueléticas/citologia , Obesidade/etiologia , Obesidade/genética , Oxirredução , Fosforilação Oxidativa , Consumo de Oxigênio , Fatores de Transcrição/genética , Aumento de Peso , Dedos de ZincoRESUMO
Restriction of HIV-1 in myeloid-lineage cells is attributed in part to the nucleotidase activity of the SAM-domain and HD-domain containing protein (SAMHD1), which depletes free nucleotides, blocking reverse transcription. In the same cells, the Vpx protein of HIV-2 and most SIVs counteracts SAMHD1. Both Type I and II interferons may stimulate SAMHD1 transcription. The contributions of SAMHD1 to retroviral restriction in the central nervous system (CNS) have been the subject of limited study. We hypothesized that SAMHD1 would respond to interferon in the SIV-infected CNS but would not control virus due to SIV Vpx. Accordingly, we investigated SAMHD1 transcript abundance and association with the Type I interferon response in an SIV model. SAMHD1 transcript levels were IFN responsive, increasing during acute phase infection and decreasing during a more quiescent phase, but generally remaining elevated at all post-infection time points. In vitro, SAMHD1 transcript was abundant in macaque astrocytes and further induced by Type I interferon, while IFN produced a weaker response in the more permissive environment of the macrophage. We cannot rule out a contribution of SAMHD1 to retroviral restriction in relatively non-permissive CNS cell types. We encourage additional research in this area, particularly in the context of HIV-1 infection.
Assuntos
Viroses do Sistema Nervoso Central/metabolismo , Macrófagos/metabolismo , Proteínas Monoméricas de Ligação ao GTP/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/metabolismo , Regulação para Cima/imunologia , Carga Viral/imunologia , Animais , Viroses do Sistema Nervoso Central/imunologia , Viroses do Sistema Nervoso Central/virologia , Feminino , Interferon Tipo I/biossíntese , Interferon Tipo I/imunologia , Macaca nemestrina , Macrófagos/imunologia , Macrófagos/virologia , Masculino , Proteínas Monoméricas de Ligação ao GTP/biossíntese , Síndrome de Imunodeficiência Adquirida dos Símios/metabolismo , Vírus da Imunodeficiência Símia/imunologiaRESUMO
BACKGROUND: The transforming growth factor ß (TGF-ß) signaling pathways modulate skeletal muscle growth, regeneration, and cellular metabolism. Several recent gene expression studies have shown that inhibition of myostatin and TGF-ß1 signaling consistently leads to a significant reduction in expression of Mss51, also named Zmynd17. The function of mammalian Mss51 is unknown although a putative homolog in yeast is a mitochondrial translational activator. OBJECTIVE: The objective of this work was to characterize mammalian Mss51. METHODS: Quantitative RT-PCR and immunoblot of subcellular fractionation were used to determine expression patterns and localization of Mss51. The CRISPR/Cas9 system was used to reduce expression of Mss51 in C2C12 myoblasts and the function of Mss51 was evaluated in assays of proliferation, differentiation and cellular metabolism. RESULTS: Mss51 was predominantly expressed in skeletal muscle and in those muscles dominated by fast-twitch fibers. In vitro, its expression was upregulated upon differentiation of C2C12 myoblasts into myotubes. Expression of Mss51 was modulated in response to altered TGF-ß family signaling. In human muscle, Mss51 localized to the mitochondria. Its genetic disruption resulted in increased levels of cellular ATP, ß-oxidation, glycolysis, and oxidative phosphorylation. CONCLUSIONS: Mss51 is a novel, skeletal muscle-specific gene and a key target of myostatin and TGF-ß1 signaling. Unlike myostatin, TGF-ß1 and IGF-1, Mss51 does not regulate myoblast proliferation or differentiation. Rather, Mss51 appears to be one of the effectors of these growth factors on metabolic processes including fatty acid oxidation, glycolysis and oxidative phosphorylation.
RESUMO
Papillomaviruses are a family of nonenveloped DNA viruses that infect the skin or mucosa of their vertebrate hosts. The viral life cycle is closely tied to the differentiation of infected keratinocytes. Papillomavirus virions are released into the environment through a process known as desquamation, in which keratinocytes lose structural integrity prior to being shed from the surface of the skin. During this process, virions are exposed to an increasingly oxidative environment, leading to their stabilization through the formation of disulfide cross-links between neighboring molecules of the major capsid protein, L1. We used time-lapse cryo-electron microscopy and image analysis to study the maturation of HPV16 capsids assembled in mammalian cells and exposed to an oxidizing environment after cell lysis. Initially, the virion is a loosely connected procapsid that, under in vitro conditions, condenses over several hours into the more familiar 60-nm-diameter papillomavirus capsid. In this process, the procapsid shrinks by ~5% in diameter, its pentameric capsomers change in structure (most markedly in the axial region), and the interaction surfaces between adjacent capsomers are consolidated. A C175S mutant that cannot achieve normal inter-L1 disulfide cross-links shows maturation-related shrinkage but does not achieve the fully condensed 60-nm form. Pseudoatomic modeling based on a 9-Å resolution reconstruction of fully mature capsids revealed C-terminal disulfide-stabilized "suspended bridges" that form intercapsomeric cross-links. The data suggest a model in which procapsids exist in a range of dynamic intermediates that can be locked into increasingly mature configurations by disulfide cross-linking, possibly through a Brownian ratchet mechanism. Importance: Human papillomaviruses (HPVs) cause nearly all cases of cervical cancer, a major fraction of cancers of the penis, vagina/vulva, anus, and tonsils, and genital and nongenital warts. HPV types associated with a high risk of cancer, such as HPV16, are generally transmitted via sexual contact. The nonenveloped virion of HPVs shows a high degree of stability, allowing the virus to persist in an infectious form in environmental fomites. In this study, we used cryo-electron microscopy to elucidate the structure of the HPV16 capsid at different stages of maturation. The fully mature capsid adopts a rigid, highly regular structure stabilized by intermolecular disulfide bonds. The availability of a pseudoatomic model of the fully mature HPV16 virion should help guide understanding of antibody responses elicited by HPV capsid-based vaccines.
Assuntos
Proteínas do Capsídeo/ultraestrutura , Papillomavirus Humano 16/crescimento & desenvolvimento , Papillomavirus Humano 16/ultraestrutura , Linhagem Celular , Microscopia Crioeletrônica , Humanos , Estrutura Secundária de Proteína , Vírion/ultraestruturaRESUMO
Mounting evidence indicates that Merkel cell polyomavirus (MCV), a circular double-stranded DNA virus, is a causal factor underlying a highly lethal form of skin cancer known as Merkel cell carcinoma. To explore the possibility that MCV and other polyomaviruses commonly inhabit healthy human skin, we developed an improved rolling circle amplification (RCA) technique to isolate circular DNA viral genomes from human skin swabs. Complete MCV genomes were recovered from 40% of healthy adult volunteers tested, providing full-length, apparently wild-type cloned MCV genomes. RCA analysis also identified two previously unknown polyomavirus species that we name human polyomavirus-6 (HPyV6) and HPyV7. Biochemical experiments show that polyomavirus DNA is shed from the skin in the form of assembled virions. A pilot serological study indicates that infection or coinfection with these three skin-tropic polyomaviruses is very common. Thus, at least three polyomavirus species are constituents of the human skin microbiome.