RESUMEN
Depolarizing sodium (Na+) leak currents carried by the NALCN channel regulate the resting membrane potential of many neurons to modulate respiration, circadian rhythm, locomotion and pain sensitivity1-8. NALCN requires FAM155A, UNC79 and UNC80 to function, but the role of these auxiliary subunits is not understood3,7,9-12. NALCN, UNC79 and UNC80 are essential in rodents2,9,13, and mutations in human NALCN and UNC80 cause severe developmental and neurological disease14,15. Here we determined the structure of the NALCN channelosome, an approximately 1-MDa complex, as fundamental aspects about the composition, assembly and gating of this channelosome remain obscure. UNC79 and UNC80 are massive HEAT-repeat proteins that form an intertwined anti-parallel superhelical assembly, which docks intracellularly onto the NALCN-FAM155A pore-forming subcomplex. Calmodulin copurifies bound to the carboxy-terminal domain of NALCN, identifying this region as a putative modulatory hub. Single-channel analyses uncovered a low open probability for the wild-type complex, highlighting the tightly closed S6 gate in the structure, and providing a basis to interpret the altered gating properties of disease-causing variants. Key constraints between the UNC79-UNC80 subcomplex and the NALCN DI-DII and DII-DIII linkers were identified, leading to a model of channelosome gating. Our results provide a structural blueprint to understand the physiology of the NALCN channelosome and a template for drug discovery to modulate the resting membrane potential.
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Canales Iónicos , Proteínas de la Membrana , Secuencias de Aminoácidos , Calmodulina , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Humanos , Activación del Canal Iónico , Canales Iónicos/química , Canales Iónicos/metabolismo , Potenciales de la Membrana , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Neuronas/metabolismo , Sodio/metabolismoRESUMEN
Engineered destruction of target proteins by recruitment to the cell's degradation machinery has emerged as a promising strategy in drug discovery. The majority of molecules that facilitate targeted degradation do so via a select number of ubiquitin ligases, restricting this therapeutic approach to tissue types that express the requisite ligase. Here, we describe a new strategy of targeted protein degradation through direct substrate recruitment to the 26S proteasome. The proteolytic complex is essential and abundantly expressed in all cells; however, proteasomal ligands remain scarce. We identify potent peptidic macrocycles that bind directly to the 26S proteasome subunit PSMD2, with a 2.5-Å-resolution cryo-electron microscopy complex structure revealing a binding site near the 26S pore. Conjugation of this macrocycle to a potent BRD4 ligand enabled generation of chimeric molecules that effectively degrade BRD4 in cells, thus demonstrating that degradation via direct proteasomal recruitment is a viable strategy for targeted protein degradation.
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Proteínas Nucleares , Factores de Transcripción , Proteínas Nucleares/metabolismo , Microscopía por Crioelectrón , Factores de Transcripción/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
The movement of core-lipopolysaccharide across the inner membrane of Gram-negative bacteria is catalysed by an essential ATP-binding cassette transporter, MsbA. Recent structures of MsbA and related transporters have provided insights into the molecular basis of active lipid transport; however, structural information about their pharmacological modulation remains limited. Here we report the 2.9 Å resolution structure of MsbA in complex with G907, a selective small-molecule antagonist with bactericidal activity, revealing an unprecedented mechanism of ABC transporter inhibition. G907 traps MsbA in an inward-facing, lipopolysaccharide-bound conformation by wedging into an architecturally conserved transmembrane pocket. A second allosteric mechanism of antagonism occurs through structural and functional uncoupling of the nucleotide-binding domains. This study establishes a framework for the selective modulation of ABC transporters and provides rational avenues for the design of new antibiotics and other therapeutics targeting this protein family.
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Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Transportadoras de Casetes de Unión a ATP/química , Antibacterianos/química , Antibacterianos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Quinolinas/química , Quinolinas/farmacología , Transportadoras de Casetes de Unión a ATP/metabolismo , Regulación Alostérica/efectos de los fármacos , Proteínas Bacterianas/metabolismo , Sitios de Unión/efectos de los fármacos , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Escherichia coli/química , Hidrocarburos/química , Hidrocarburos/metabolismo , Lipopolisacáridos/química , Lipopolisacáridos/metabolismo , Lipopolisacáridos/farmacología , Modelos Moleculares , Dominios Proteicos/efectos de los fármacosRESUMEN
Nanolipoprotein particles (NLPs), a lipid bilayer-based nanoparticle platform, have recently been developed for in vivo delivery of a variety of molecules of therapeutic interest, but their potential to deliver Fabs with valencies that exceed those of current multivalent formats has not yet been evaluated. Here we describe the development, optimization, and characterization of Fab-NLP conjugates. NLPs were generated with maleimide reactive lipids for conjugation to a Fab with a C-terminal cysteine. Of note, maleimide reactive lipids were shown to conjugate to the apolipoprotein when the NLPs were assembled at pH 7.4. However, this undesirable reaction was not observed when assembled at pH 6. Site-specific Fab conjugation conditions were then optimized, and conjugation of up to 30 Fab per NLP was demonstrated. Interestingly, although conjugation of higher numbers of Fabs had a significant impact on NLP molecular weight, only a minimal impact on NLP hydrodynamic radius was observed, indicating that particle size is largely dictated by the discoidal shape of the NLP. Fab-NLP viscosity and its stability upon lyophilization were also evaluated as an assessment of the manufacturability of the Fab-NLP. Significantly higher Fab concentrations were achieved with the Fab-NLP conjugates relative to another multivalent format (Fab-PEG conjugates). Fab conjugation to the NLP was also not found to have an impact on Fab activity in both an inhibitory and agonist setting. Finally, the stability of the Fab-NLP conjugates was evaluated in 50% serum and Fab-NLPs demonstrated increased stability, with >63% of Fab-NLP remaining intact after 24 h at Fab per particle ratios of 7 or greater. Our findings suggest Fab-NLPs are a promising platform for the targeted delivery of Fabs in a multivalent format and are compatible with established manufacturing processes.
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Fragmentos Fab de Inmunoglobulinas/química , Lipoproteínas/química , Nanoestructuras/química , Sistemas de Liberación de Medicamentos , Fragmentos Fab de Inmunoglobulinas/farmacología , Maleimidas/química , ReologíaRESUMEN
Ubiquitin is a highly conserved eukaryotic protein that interacts with a diverse set of partners to act as a cellular signaling hub. Ubiquitin's conformational flexibility has been postulated to underlie its multifaceted recognition. Here we use computational and library-based means to interrogate core mutations that modulate the conformational dynamics of human ubiquitin. These ubiquitin variants exhibit increased affinity for the USP14 deubiquitinase, with concomitantly reduced affinity for other deubiquitinases. Strikingly, the kinetics of conformational motion are dramatically slowed in these variants without a detectable change in either the ground state fold or excited state population. These variants can be ligated into substrate-linked chains in vitro and in vivo but cannot solely support growth in eukaryotic cells. Proteomic analyses reveal nearly identical interaction profiles between WT ubiquitin and the variants but identify a small subset of altered interactions. Taken together, these results show that conformational dynamics are critical for ubiquitin-deubiquitinase interactions and imply that the fine tuning of motion has played a key role in the evolution of ubiquitin as a signaling hub.
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Endopeptidasas/metabolismo , Transducción de Señal , Ubiquitina/metabolismo , Secuencia de Aminoácidos , Endopeptidasas/química , Modelos Moleculares , Unión Proteica , Conformación Proteica , Ubiquitina/químicaRESUMEN
INTRODUCTION: Breast cancer cells display preferences for specific metastatic sites including the bone, lung and liver. Metastasis is a complex process that relies, in part, on interactions between disseminated cancer cells and resident/infiltrating stromal cells that constitute the metastatic microenvironment. Distinct immune infiltrates can either impair the metastatic process or conversely, assist in the seeding, colonization and growth of disseminated cancer cells. METHODS: Using in vivo selection approaches, we previously isolated 4T1-derived breast cancer cells that preferentially metastasize to these organs and tissues. In this study, we examined whether the propensity of breast cancer cells to metastasize to the lung, liver or bone is associated with and dependent on distinct patterns of immune cell infiltration. Immunohistocytochemistry and immunohistofluorescence approaches were used to quantify innate immune cell infiltrates within distinct metastases and depletion of Gr1+ (Ly-6C and Ly-6G) or specifically Ly-6G+ cells was performed to functionally interrogate the role of Ly-6G+ infiltrates in promoting metastasis to these organs. RESULTS: We show that T lymphocytes (CD3+), myeloid-derived (Gr-1+) cells and neutrophils (Ly-6G+ or NE+) exhibit the most pronounced recruitment in lung and liver metastases, with markedly less recruitment within bone metastatic lesions. Interestingly, these infiltrating cell populations display different patterns of localization within soft tissue metastases. T lymphocytes and granulocytic immune infiltrates are localized around the periphery of liver metastases whereas they were dispersed throughout the lung metastases. Furthermore, Gr-1+ cell-depletion studies demonstrate that infiltrating myeloid-derived cells are essential for the formation of breast cancer liver metastases but dispensable for metastasis to the lung and bone. A specific role for the granulocytic component of the innate immune infiltrate was revealed through Ly-6G+ cell-depletion experiments, which resulted in significantly impaired formation of liver metastases. Finally, we demonstrate that the CD11b+/Ly-6G+ neutrophils that infiltrate and surround the liver metastases are polarized toward an N2 phenotype, which have previously been shown to enhance tumor growth and metastasis. CONCLUSIONS: Our results demonstrate that the liver-metastatic potential of breast cancer cells is heavily reliant on interactions with infiltrating Ly-6G+ cells within the liver microenvironment.
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Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Granulocitos/inmunología , Granulocitos/patología , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/secundario , Biomarcadores , Neoplasias Óseas/inmunología , Neoplasias Óseas/patología , Neoplasias Óseas/secundario , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Quimiocinas/genética , Quimiocinas/metabolismo , Análisis por Conglomerados , Progresión de la Enfermedad , Femenino , Perfilación de la Expresión Génica/métodos , Granulocitos/metabolismo , Humanos , Inmunohistoquímica , Inmunofenotipificación , Neoplasias Hepáticas/patología , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/secundario , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/patología , Infiltración Neutrófila/inmunología , Transducción de Señal , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Transcriptoma , Microambiente Tumoral/inmunologíaRESUMEN
Histone deacetylases (HDACs) are critical in the control of gene expression, and dysregulation of their activity has been implicated in a broad range of diseases, including cancer, cardiovascular, and neurological diseases. HDAC inhibitors (HDACi) employing different zinc chelating functionalities such as hydroxamic acids and benzamides have shown promising results in cancer therapy. Although it has also been suggested that HDACi with increased isozyme selectivity and potency may broaden their clinical utility and minimize side effects, the translation of this idea to the clinic remains to be investigated. Moreover, a detailed understanding of how HDACi with different pharmacological properties affect biological functions in vitro and in vivo is still missing. Here, we show that a panel of benzamide-containing HDACi are slow tight-binding inhibitors with long residence times unlike the hydroxamate-containing HDACi vorinostat and trichostatin-A. Characterization of changes in H2BK5 and H4K14 acetylation following HDACi treatment in the neuroblastoma cell line SH-SY5Y revealed that the timing and magnitude of histone acetylation mirrored both the association and dissociation kinetic rates of the inhibitors. In contrast, cell viability and microarray gene expression analysis indicated that cell death induction and changes in transcriptional regulation do not correlate with the dissociation kinetic rates of the HDACi. Therefore, our study suggests that determining how the selective and kinetic inhibition properties of HDACi affect cell function will help to evaluate their therapeutic utility.
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Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Inhibidores de Histona Desacetilasas/química , Histonas/química , Acetilación , Benzamidas/química , Unión Competitiva , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , Ácidos Hidroxámicos/química , Concentración 50 Inhibidora , Cinética , Análisis de Secuencia por Matrices de Oligonucleótidos , Unión Proteica , Piridinas/química , Transcripción Genética , VorinostatRESUMEN
The caspases are a family of cytosolic proteases with essential roles in inflammation and apoptosis. Drug discovery efforts have focused on developing molecules directed against the active sites of caspases, but this approach has proved challenging and has not yielded any approved therapeutics. Here we describe a new strategy for generating inhibitors of caspase-6, a potential therapeutic target in neurodegenerative disorders, by screening against its zymogen form. Using phage display to discover molecules that bind the zymogen, we report the identification of a peptide that specifically impairs the function of caspase-6 in vitro and in neuronal cells. Remarkably, the peptide binds at a tetramerization interface that is uniquely present in zymogen caspase-6, rather than binding into the active site, and acts via a new allosteric mechanism that promotes caspase tetramerization. Our data illustrate that screening against the zymogen holds promise as an approach for targeting caspases in drug discovery.
Asunto(s)
Biopolímeros/metabolismo , Caspasa 6/metabolismo , Precursores Enzimáticos/metabolismo , Péptidos/metabolismo , Regulación Alostérica , Línea Celular Tumoral , Ensayo de Inmunoadsorción Enzimática , Humanos , Péptidos/química , Unión ProteicaRESUMEN
Metabolic rewiring is essential for tumor growth and progression to metastatic disease, yet little is known regarding how cancer cells modify their acquired metabolic programs in response to different metastatic microenvironments. We have previously shown that liver-metastatic breast cancer cells adopt an intrinsic metabolic program characterized by increased HIF-1α activity and dependence on glycolysis. Here, we confirm by in vivo stable isotope tracing analysis (SITA) that liver-metastatic breast cancer cells retain a glycolytic profile when grown as mammary tumors or liver metastases. However, hepatic metastases exhibit unique metabolic adaptations including elevated expression of genes involved in glutathione (GSH) biosynthesis and reactive oxygen species (ROS) detoxification when compared to mammary tumors. Accordingly, breast-cancer-liver-metastases exhibited enhanced de novo GSH synthesis. Confirming their increased capacity to mitigate ROS-mediated damage, liver metastases display reduced levels of 8-Oxo-2'-deoxyguanosine. Depletion of the catalytic subunit of the rate-limiting enzyme in glutathione biosynthesis, glutamate-cysteine ligase (GCLC), strongly reduced the capacity of breast cancer cells to form liver metastases, supporting the importance of these distinct metabolic adaptations. Loss of GCLC also affected the early steps of the metastatic cascade, leading to decreased numbers of circulating tumor cells (CTCs) and impaired metastasis to the liver and the lungs. Altogether, our results indicate that GSH metabolism could be targeted to prevent the dissemination of breast cancer cells.
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Neoplasias de la Mama , Glutamato-Cisteína Ligasa , Glutatión , Homeostasis , Neoplasias Hepáticas , Oxidación-Reducción , Especies Reactivas de Oxígeno , Femenino , Neoplasias de la Mama/patología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/genética , Humanos , Glutatión/metabolismo , Animales , Especies Reactivas de Oxígeno/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/secundario , Neoplasias Hepáticas/genética , Ratones , Línea Celular Tumoral , Glutamato-Cisteína Ligasa/metabolismo , Glutamato-Cisteína Ligasa/genética , Glucólisis , Metástasis de la Neoplasia , Regulación Neoplásica de la Expresión Génica , Microambiente TumoralRESUMEN
The drug discovery landscape is ever-evolving and constantly demands revolutionary technology advancements in protein expression and production laboratories. We have built a higher-throughput mid-scale semi-automated protein expression and screening platform to accelerate drug discovery research. The workflow described here enables comprehensive expression and purification screening assessment of challenging or difficult-to-express recombinant proteins in a fast and efficient manner by delivering small but sufficient amounts of high-quality proteins. The platform has been implemented for a wide range of applications that include identification of optimal constructs and chaperones for poorly expressing proteins, assessment of co-expression partners for expressing stable multiprotein complexes, and suitable buffer/additive screening for insoluble or aggregation-prone proteins. The approach allows parallel expression, purification, and characterization of 24 different samples using co-infection or a polycistronic approach in insect cells and enables parallel testing of multiple parameters to improve protein yields. The strategy has been successfully adopted for screening intracellular and secreted proteins in Escherichia coli, mammalian transient expression, and baculovirus expression vector systems. Proteins purified from this platform are used for several structural and functional screens, such as negative staining, biochemical activity assays, mass spectrometry, surface plasmon resonance, and DNA-encoded chemical library screens. In this article, for simplicity, we have focused on detailed expression and purification screening of intracellular protein complexes from insect cells. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Baculovirus generation via homologous recombination Support Protocol 1: Anti-glycoprotein 64 antibody assay Basic Protocol 2: Generation of insect cell biomass expressing target protein(s) Basic Protocol 3: Mid-scale affinity purification Support Protocol 2: Automated method for affinity purification on Hamilton STAR Basic Protocol 4: Size exclusion chromatography Support Protocol 3: Chromeleon 7 operation on Vanquish Duo.
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Acetaminofén , Aspirina , Animales , Baculoviridae , Bioensayo , Descubrimiento de Drogas , Escherichia coli , MamíferosRESUMEN
The C-C motif chemokine receptor 8 (CCR8) is a class A G-protein coupled receptor that has emerged as a promising therapeutic target in cancer. Targeting CCR8 with an antibody has appeared to be an attractive therapeutic approach, but the molecular basis for chemokine-mediated activation and antibody-mediated inhibition of CCR8 are not fully elucidated. Here, we obtain an antagonist antibody against human CCR8 and determine structures of CCR8 in complex with either the antibody or the endogenous agonist ligand CCL1. Our studies reveal characteristic antibody features allowing recognition of the CCR8 extracellular loops and CCL1-CCR8 interaction modes that are distinct from other chemokine receptor - ligand pairs. Informed by these structural insights, we demonstrate that CCL1 follows a two-step, two-site binding sequence to CCR8 and that antibody-mediated inhibition of CCL1 signaling can occur by preventing the second binding event. Together, our results provide a detailed structural and mechanistic framework of CCR8 activation and inhibition that expands our molecular understanding of chemokine - receptor interactions and offers insight into the development of therapeutic antibodies targeting chemokine GPCRs.
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Quimiocinas CC , Receptores de Quimiocina , Humanos , Quimiocinas CC/metabolismo , Quimiocinas CC/farmacología , Receptores CCR8/genética , Ligandos , Quimiocina CCL1/metabolismo , Receptores de Quimiocina/genética , AnticuerposRESUMEN
Unlike classical voltage-gated sodium (NaV) channels, NaX has been characterized as a voltage-insensitive, tetrodotoxin-resistant, sodium (Na+)-activated channel involved in regulating Na+ homeostasis. However, NaX remains refractory to functional characterization in traditional heterologous systems. Here, to gain insight into its atypical physiology, we determine structures of the human NaX channel in complex with the auxiliary ß3-subunit. NaX reveals structural alterations within the selectivity filter, voltage sensor-like domains, and pore module. We do not identify an extracellular Na+-sensor or any evidence for a Na+-based activation mechanism in NaX. Instead, the S6-gate remains closed, membrane lipids fill the central cavity, and the domain III-IV linker restricts S6-dilation. We use protein engineering to identify three pore-wetting mutations targeting the hydrophobic S6-gate that unlock a robust voltage-insensitive leak conductance. This constitutively active NaX-QTT channel construct is non-selective among monovalent cations, inhibited by extracellular calcium, and sensitive to classical NaV channel blockers, including tetrodotoxin. Our findings highlight a functional diversity across the NaV channel scaffold, reshape our understanding of NaX physiology, and provide a template to demystify recalcitrant ion channels.
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Calcio , Sodio , Calcio/metabolismo , Cationes , Humanos , Sodio/metabolismo , Tetrodotoxina/farmacologíaRESUMEN
The epidermis is a barrier that prevents water loss while keeping harmful substances from penetrating the host. The impermeable cornified layer of the stratum corneum is maintained by balancing continuous turnover driven by epidermal basal cell proliferation, suprabasal cell differentiation, and corneal shedding. The epidermal desquamation process is tightly regulated by balance of the activities of serine proteases of the Kallikrein-related peptidases (KLK) family and their cognate inhibitor lymphoepithelial Kazal type-related inhibitor (LEKTI), which is encoded by the serine peptidase inhibitor Kazal type 5 gene. Imbalance of proteolytic activity caused by a deficiency of LEKTI leads to excessive desquamation due to increased activities of KLK5, KLK7, and KLK14 and results in Netherton syndrome (NS), a debilitating condition with an unmet clinical need. Increased activity of KLKs may also be pathological in other dermatoses such as atopic dermatitis (AD). Here, we describe the discovery of inhibitory antibodies against murine KLK5 and KLK7 that could compensate for the deficiency of LEKTI in NS. These antibodies are protective in mouse models of NS and AD and, when combined, promote improved skin barrier integrity and reduced inflammation. To translate these findings, we engineered a humanized bispecific antibody capable of potent inhibition of human KLK5 and KLK7. A crystal structure of KLK5 bound to the inhibitory Fab revealed that the antibody binds distal to its active site and uses a relatively unappreciated allosteric inhibition mechanism. Treatment with the bispecific anti-KLK5/7 antibody represents a promising therapy for clinical development in NS and other inflammatory dermatoses.
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Dermatitis Atópica , Síndrome de Netherton , Enfermedades de la Piel , Ratones , Humanos , Animales , Síndrome de Netherton/genética , Síndrome de Netherton/metabolismo , Síndrome de Netherton/patología , Dermatitis Atópica/patología , Inhibidor de Serinpeptidasas Tipo Kazal-5/metabolismo , Epidermis/patología , Enfermedades de la Piel/metabolismo , Anticuerpos/metabolismo , Calicreínas/metabolismoRESUMEN
Wnt/beta-catenin signaling is initiated at the cell surface by association of secreted Wnt with its receptors Frizzled (Fz) and low density lipoprotein receptor-related protein 5/6 (LRP5/6). The study of these molecular interactions has been a significant technical challenge because the proteins have been inaccessible in sufficient purity and quantity. In this report we describe insect cell expression and purification of soluble mouse Fz8 cysteine-rich domain and human LRP6 extracellular domain and show that they inhibit Wnt/beta-catenin signaling in cellular assays. We determine the binding affinities of Wnts and Dickkopf 1 (Dkk1) to the relevant co-receptors and reconstitute in vitro the Fz8 CRD.Wnt3a.LRP6 signaling complex. Using purified fragments of LRP6, we further show that Wnt3a binds to a region including only the third and fourth beta-propeller domains of LRP6 (E3E4). Surprisingly, we find that Wnt9b binds to a different part of the LRP6 extracellular domain, E1E2, and we demonstrate that Wnt3a and Wnt9b can bind to LRP6 simultaneously. Dkk1 binds to both E1E2 and E3E4 fragments and competes with both Wnt3a and Wnt9b for binding to LRP6. The existence of multiple, independent Wnt binding sites on the LRP6 co-receptor suggests new possibilities for the architecture of Wnt signaling complexes and a model for broad-spectrum inhibition of Wnt/beta-catenin signaling by Dkk1.
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Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas Relacionadas con Receptor de LDL/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/fisiología , Proteínas Wnt/metabolismo , Animales , Sitios de Unión , Relación Dosis-Respuesta a Droga , Humanos , Concentración 50 Inhibidora , Insectos , Cinética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad , Ratones , Unión Proteica , Estructura Terciaria de Proteína , Proteína Wnt3 , Proteína Wnt3A , beta Catenina/metabolismoRESUMEN
To address devastating environmental crises and to improve human well-being, China has been implementing a number of national policies on payments for ecosystem services. Two of them, the Natural Forest Conservation Program (NFCP) and the Grain to Green Program (GTGP), are among the biggest programs in the world because of their ambitious goals, massive scales, huge payments, and potentially enormous impacts. The NFCP conserves natural forests through logging bans and afforestation with incentives to forest enterprises, whereas the GTGP converts cropland on steep slopes to forest and grassland by providing farmers with grain and cash subsidies. Overall ecological effects are beneficial, and socioeconomic effects are mostly positive. Whereas there are time lags in ecological effects, socioeconomic effects are more immediate. Both the NFCP and the GTGP also have global implications because they increase vegetative cover, enhance carbon sequestration, and reduce dust to other countries by controlling soil erosion. The future impacts of these programs may be even bigger. Extended payments for the GTGP have recently been approved by the central government for up to 8 years. The NFCP is likely to follow suit and receive renewed payments. To make these programs more effective, we recommend systematic planning, diversified funding, effective compensation, integrated research, and comprehensive monitoring. Effective implementation of these programs can also provide important experiences and lessons for other ecosystem service payment programs in China and many other parts of the world.
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Ecosistema , Ambiente , Factores Socioeconómicos , China , Conservación de los Recursos Naturales/economía , MotivaciónRESUMEN
Primary cutaneous mucinous carcinoma is a neoplasm of sweat gland origin. Optimal management guidelines have not been established for this rare tumor. It is treated most commonly by traditional excision and more recently by Mohs micrographic surgery in an effort to decrease its recurrence rate. We report a case of primary cutaneous mucinous carcinoma with multiple recurrences and metastases following 3 excisions and 2 Mohs procedures, highlighting the potential difficulty in treating this cancer and suggesting the need for a more effective treatment approach.
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Adenocarcinoma Mucinoso/patología , Cirugía de Mohs/métodos , Neoplasias Cutáneas/patología , Adenocarcinoma Mucinoso/cirugía , Humanos , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia , Recurrencia Local de Neoplasia , Reoperación , Neoplasias Cutáneas/cirugíaRESUMEN
Despite the need to monitor the impact of Cancer Immunotherapy (CI)/Immuno-Oncology (IO) therapeutics on neoantigen-specific T-cell responses, very few clinical programs incorporate this aspect of immune monitoring due to the challenges in high-throughput (HTP) generation of Major Histocompatibility Complex Class I (MHCI) tetramers across a wide range of HLA alleles. This limitation was recently addressed through the development of MHCI complexes with peptides containing a nonnatural UV cleavable amino acid (conditional MHCI ligands) that enabled HTP peptide exchange upon UV exposure. Despite this advancement, the number of alleles with known conditional MHCI ligands is limited. We developed a novel workflow to enable identification and validation of conditional MHCI ligands across a range of HLA alleles. First, known peptide binders were screened via an enzyme-linked immunosorbent assay (ELISA) assay. Conditional MHCI ligands were designed using the highest-performing peptides and evaluated in the same ELISA assay. The top performers were then selected for scale-up production. Next-generation analytical techniques (LC/MS, SEC-MALS, and 2D LC/MS) were used to characterize the complex after refolding with the conditional MHCI ligands. Finally, we used 2D LC/MS to evaluate peptide exchange with these scaled-up conditional MHCI complexes after UV exposure with validated peptide binders. Successful peptide exchange was observed for all conditional MHCI ligands upon UV exposure, validating our screening approach. This approach has the potential to be broadly applied and enable HTP generation of MHCI monomers and tetramers across a wider range of HLA alleles, which could be critical to enabling the use of MHCI tetramers to monitor neoantigen-specific T-cells in the clinic.
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Antígenos de Histocompatibilidad Clase I/química , Péptidos/química , Humanos , LigandosRESUMEN
Nanolipoprotein particles (NLPs) have been evaluated as an in vivo delivery vehicle for a variety of molecules of therapeutic interest. However, delivery of peptide-like drugs in combination with therapeutic Fabs has not yet been evaluated. In this study, we describe the development and characterization of cystine-knot peptide (CKP)-containing NLPs and Fab-CKP-NLP conjugates. CKPs were incorporated into NLPs using a self-assembly strategy. The trypsin inhibitor EETI-II, a model CKP, was produced with a C16 fatty acyl chain to enable incorporation of the CKP into the lipid bilayer core during NLP assembly. The CKP-NLP retained trypsin inhibitory function although the overall activity was reduced by â¼5 fold compared to free CKP, which was presumably due to steric hindrance. The NLP platform was also shown to accommodate up to â¼60 CKP molecules. Moreover, the stability of the CKP-NLP was comparable to the NLP control, displaying a relatively short half-life (â¼1 h) in 50% serum at 37 °C. Therapeutic Fabs were also loaded onto the CKP-NLP by introducing thiol-reactive lipids that would undergo a covalent reaction with the Fab. Using this strategy, Fab loading could be reliably controlled from 1-50 Fabs per CKP-NLP and was found to be independent of CKP density. Surprisingly, Fab incorporation into CKP-NLPs led to a substantial improvement in NLP stability (half-life > 24 h) at 37 °C; also, there was no reduction in CKP activity in the Fab-CKP-NLP conjugates compared to CKP-NLPs. Altogether, our data demonstrate the potential of NLPs as a promising platform for the targeted or multidrug delivery of peptide-based drug candidates in combination with Fabs.
RESUMEN
The TRPA1 ion channel is activated by electrophilic compounds through the covalent modification of intracellular cysteine residues. How non-covalent agonists activate the channel and whether covalent and non-covalent agonists elicit the same physiological responses are not understood. Here, we report the discovery of a non-covalent agonist, GNE551, and determine a cryo-EM structure of the TRPA1-GNE551 complex, revealing a distinct binding pocket and ligand-interaction mechanism. Unlike the covalent agonist allyl isothiocyanate, which elicits channel desensitization, tachyphylaxis, and transient pain, GNE551 activates TRPA1 into a distinct conducting state without desensitization and induces persistent pain. Furthermore, GNE551-evoked pain is relatively insensitive to antagonist treatment. Thus, we demonstrate the biased agonism of TRPA1, a finding that has important implications for the discovery of effective drugs tailored to different disease etiologies.
Asunto(s)
Dimensión del Dolor/métodos , Canal Catiónico TRPA1/agonistas , Canal Catiónico TRPA1/metabolismo , Secuencia de Aminoácidos , Animales , Femenino , Células HEK293 , Humanos , Ligandos , Masculino , Dimensión del Dolor/efectos de los fármacos , Estructura Secundaria de Proteína , Ratas , Ratas Sprague-Dawley , Ratas Transgénicas , Canal Catiónico TRPA1/químicaRESUMEN
Despite the development of effective therapies, a substantial proportion of asthmatics continue to have uncontrolled symptoms, airflow limitation, and exacerbations. Transient receptor potential cation channel member A1 (TRPA1) agonists are elevated in human asthmatic airways, and in rodents, TRPA1 is involved in the induction of airway inflammation and hyperreactivity. Here, the discovery and early clinical development of GDC-0334, a highly potent, selective, and orally bioavailable TRPA1 antagonist, is described. GDC-0334 inhibited TRPA1 function on airway smooth muscle and sensory neurons, decreasing edema, dermal blood flow (DBF), cough, and allergic airway inflammation in several preclinical species. In a healthy volunteer Phase 1 study, treatment with GDC-0334 reduced TRPA1 agonist-induced DBF, pain, and itch, demonstrating GDC-0334 target engagement in humans. These data provide therapeutic rationale for evaluating TRPA1 inhibition as a clinical therapy for asthma.