RESUMO
Dengue viruses (DENV serotypes 1-4) and Zika virus (ZIKV) are related flaviviruses that continue to be a public health concern, infecting hundreds of millions of people annually. The traditional live-attenuated virus vaccine approach has been challenging for the four DENV serotypes because of the need to achieve balanced replication of four independent vaccine components. Subunit vaccines represent an alternative approach that may circumvent problems inherent with live-attenuated DENV vaccines. In mature virus particles, the envelope (E) protein forms a homodimer that covers the surface of the virus and is the major target of neutralizing antibodies. Many neutralizing antibodies bind to quaternary epitopes that span across both E proteins in the homodimer. For soluble E (sE) protein to be a viable subunit vaccine, the antigens should be easy to produce and retain quaternary epitopes recognized by neutralizing antibodies. However, WT sE proteins are primarily monomeric at conditions relevant for vaccination and exhibit low expression yields. Previously, we identified amino acid mutations that stabilize the sE homodimer from DENV2 and dramatically raise expression yields. Here, we tested whether these same mutations raise the stability of sE from other DENV serotypes and ZIKV. We show that the mutations raise thermostability for sE from all the viruses, increase production yields from 4-fold to 250-fold, stabilize the homodimer, and promote binding to dimer-specific neutralizing antibodies. Our findings suggest that these sE variants could be valuable resources in the efforts to develop effective subunit vaccines for DENV serotypes 1 to 4 and ZIKV.
Assuntos
Vírus da Dengue , Vacinas de Subunidades Antigênicas , Proteínas do Envelope Viral , Vacinas Virais , Zika virus , Anticorpos Neutralizantes , Anticorpos Antivirais , Reações Cruzadas , Dengue/prevenção & controle , Vírus da Dengue/genética , Epitopos , Humanos , Mutação , Vacinas Atenuadas , Vacinas de Subunidades Antigênicas/genética , Proteínas do Envelope Viral/genética , Vacinas Virais/genética , Zika virus/genética , Infecção por Zika virus/prevenção & controleRESUMO
The present study focused on the effect of different drying temperatures (40, 50, 60 and 70 °C) and combination of pre-treatments: potassium metabisulphite (KMS), potassium metabisulphite + Citric acid + blanching (KCB)] on functional, thermo-pasting and antioxidant properties of elephant foot yam (EFY) powder. Drying temperature and pretreatment reduces the water and oil absorption capacity, and the highest values were 2.34 g/g and 1.19 g/g for drying at 40 °C for the untreated sample, respectively. KMS pretreatment enhanced the bulk density, foaming capacity, emulsion capacity, and emulsion stability with an increase in drying temperature. Pasting temperature and viscosity decreased with an increase in drying temperature, and the maximum was observed at 40 °C for KMS pretreatment. Blanching increases the gelatinization temperature resulting in higher mid-and end-temperatures for KCB pretreatment. The antioxidant properties decreased with an increase in the drying temperature and were found to be minimal in the case of KCB treated samples.
RESUMO
Polycomb repressive complex 2 (PRC2) regulates pluripotency, differentiation, and tumorigenesis through catalysis of histone H3 lysine 27 trimethylation (H3K27me3) on chromatin. However, the mechanisms that underlie PRC2 recruitment and spreading on chromatin remain unclear. Here we report that histone H3 lysine 36 trimethylation (H3K36me3) binding activity is harbored in the Tudor motifs of PRC2-associated polycomb-like (PCL) proteins PHF1/PCL1 and PHF19/PCL3. Ectopically expressed PHF1 induced Tudor-dependent stabilization of PRC2 complexes on bulk chromatin and mediated spreading of PRC2 and H3K27me3 into H3K36me3-containing chromatin regions. In murine pluripotent stem cells, we identified coexistence of H3K36me3, H3K27me3, and PHF19/PCL3 at a subset of poised developmental genes and demonstrated that PHF19/PCL3 Tudor function is required for optimal H3K27me3 and repression of these loci. Collectively, our data suggest that PCL recognition of H3K36me3 promotes intrusion of PRC2 complexes into active chromatin regions to promote gene silencing and modulate the chromatin landscape during development.
Assuntos
Histonas/metabolismo , Lisina/metabolismo , Complexo Repressor Polycomb 2/química , Complexo Repressor Polycomb 2/metabolismo , Motivos de Aminoácidos , Animais , Calorimetria , Proteínas de Transporte/metabolismo , Diferenciação Celular/genética , Núcleo Celular/metabolismo , Sequência Conservada , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Teste de Complementação Genética , Loci Gênicos , Humanos , Metilação , Camundongos , Modelos Moleculares , Proteínas Nucleares/metabolismo , Peptídeos/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Proteínas do Grupo Polycomb , Ligação Proteica , Transporte Proteico , Relação Estrutura-Atividade , Termodinâmica , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
The gut microbiota harbor diverse ß-glucuronidase (GUS) enzymes that liberate glucuronic acid (GlcA) sugars from small-molecule conjugates and complex carbohydrates. However, only the Enterobacteriaceae family of human gut-associated Proteobacteria maintain a GUS operon under the transcriptional control of a glucuronide repressor, GusR. Despite its potential importance in Escherichia, Salmonella, Klebsiella, Shigella, and Yersinia opportunistic pathogens, the structure of GusR has not been examined. Here, we explore the molecular basis for GusR-mediated regulation of GUS expression in response to small-molecule glucuronides. Presented are 2.1-Å-resolution crystal structures of GusRs from Escherichia coli and Salmonella enterica in complexes with a glucuronide ligand. The GusR-specific DNA operator site in the regulatory region of the E. coli GUS operon is identified, and structure-guided GusR mutants pinpoint the residues essential for DNA binding and glucuronide recognition. Interestingly, the endobiotic estradiol-17-glucuronide and the xenobiotic indomethacin-acyl-glucuronide are found to exhibit markedly differential binding to these GusR orthologs. Using structure-guided mutations, we are able to transfer E. coli GusR's preferential DNA and glucuronide binding affinity to S. enterica GusR. Structures of putative GusR orthologs from GUS-encoding Firmicutes species also reveal functionally unique features of the Enterobacteriaceae GusRs. Finally, dominant-negative GusR variants are validated in cell-based studies. These data provide a molecular framework toward understanding the control of glucuronide utilization by opportunistic pathogens in the human gut.
Assuntos
Proteínas de Bactérias/genética , Enterobacteriaceae/genética , Microbioma Gastrointestinal/genética , Regulação Bacteriana da Expressão Gênica , Glucuronidase/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , DNA/química , DNA/genética , DNA/metabolismo , Enterobacteriaceae/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Reguladores/genética , Ácido Glucurônico/química , Ácido Glucurônico/metabolismo , Glucuronidase/química , Glucuronidase/metabolismo , Humanos , Mutação , Óperon/genética , Homologia de Sequência de AminoácidosRESUMO
The glycoside hydrolases encoded by the human gut microbiome play an integral role in processing a variety of exogenous and endogenous glycoconjugates. Here we present three structurally and functionally distinct ß-glucuronidase (GUS) glycoside hydrolases from a single human gut commensal microbe, Bacteroides uniformis We show using nine crystal structures, biochemical, and biophysical data that whereas these three proteins share similar overall folds, they exhibit different structural features that create three structurally and functionally unique enzyme active sites. Notably, quaternary structure plays an important role in creating distinct active site features that are hard to predict via structural modeling methods. The enzymes display differential processing capabilities toward glucuronic acid-containing polysaccharides and SN-38-glucuronide, a metabolite of the cancer drug irinotecan. We also demonstrate that GUS-specific and nonselective inhibitors exhibit varying potencies toward each enzyme. Together, these data highlight the diversity of GUS enzymes within a single Bacteroides gut commensal and advance our understanding of how structural details impact the specific roles microbial enzymes play in processing drug-glucuronide and glycan substrates.
Assuntos
Bacteroides/enzimologia , Microbioma Gastrointestinal , Glucuronidase/química , Glucuronidase/metabolismo , Isoenzimas/química , Isoenzimas/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Inibidores Enzimáticos/farmacologia , Ácido Glucárico/análogos & derivados , Glucuronidase/antagonistas & inibidores , Humanos , Conformação ProteicaRESUMO
The spread of dengue (DENV) and Zika virus (ZIKV) is a major public health concern. The primary target of antibodies that neutralize DENV and ZIKV is the envelope (E) glycoprotein, and there is interest in using soluble recombinant E (sRecE) proteins as subunit vaccines. However, the most potent neutralizing antibodies against DENV and ZIKV recognize epitopes on the virion surface that span two or more E proteins. Therefore, to create effective DENV and ZIKV vaccines, presentation of these quaternary epitopes may be necessary. The sRecE proteins from DENV and ZIKV crystallize as native-like dimers, but studies in solution suggest that these dimers are marginally stable. To better understand the challenges associated with creating stable sRecE dimers, we characterized the thermostability of sRecE proteins from ZIKV and three DENV serotypes, DENV2-4. All four proteins irreversibly unfolded at moderate temperatures (46-53 °C). At 23 °C and low micromolar concentrations, DENV2 and ZIKV were primarily dimeric, and DENV3-4 were primarily monomeric, whereas at 37 °C, all four proteins were predominantly monomeric. We further show that the dissociation constant for DENV2 dimerization is very temperature-sensitive, ranging from <1 µm at 25 °C to 50 µm at 41 °C, due to a large exothermic enthalpy of binding of -79 kcal/mol. We also found that quaternary epitope antibody binding to DENV2-4 and ZIKV sRecE is reduced at 37 °C. Our observation of reduced sRecE dimerization at physiological temperature highlights the need for stabilizing the dimer as part of its development as a subunit vaccine.
Assuntos
Vírus da Dengue/química , Multimerização Proteica , Proteínas do Envelope Viral/química , Zika virus/química , Temperatura Corporal , Dengue/virologia , Humanos , Estabilidade Proteica , Proteínas Recombinantes/química , Vacinas de Subunidades Antigênicas/química , Vacinas Virais/química , Infecção por Zika virus/virologiaRESUMO
The opportunistic bacteria of the Burkholderia cepacia complex (Bcc) are extremely pathogenic to cystic fibrosis (CF) patients, and acquisition of Bcc bacteria is associated with a significant increase in mortality. Treatment of Bcc infections is difficult because the bacteria are multidrug resistant and able to survive in biofilms. Short palate, lung, and nasal epithelial clone 1 (SPLUNC1) is an innate defense protein that is secreted by the upper airways and pharynx. While SPLUNC1 is known to have antimicrobial functions, its effects on Bcc strains are unclear. We therefore tested the hypothesis that SPLUNC1 is able to impair Bcc growth and biofilm formation. We found that SPLUNC1 exerted bacteriostatic effects against several Bcc clinical isolates, including B. cenocepacia strain J2315 (50% inhibitory concentration [IC50] = 0.28 µM), and reduced biofilm formation and attachment (IC50 = 0.11 µM). We then determined which domains of SPLUNC1 are responsible for its antimicrobial activity. Deletions of SPLUNC1's N terminus and α6 helix did not affect its function. However, deletion of the α4 helix attenuated antimicrobial activity, while the corresponding α4 peptide displayed antimicrobial activity. Chronic neutrophilia is a hallmark of CF lung disease, and neutrophil elastase (NE) cleaves SPLUNC1. However, we found that the ability of SPLUNC1 to disrupt biofilm formation was significantly potentiated by NE pretreatment. While the impact of CF on SPLUNC1-Bcc interactions is not currently known, our data suggest that understanding this interaction may have important implications for CF lung disease.
Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Complexo Burkholderia cepacia/efeitos dos fármacos , Glicoproteínas/farmacologia , Elastase de Leucócito/química , Fosfoproteínas/farmacologia , Antibacterianos/química , Antibacterianos/metabolismo , Sítios de Ligação , Biofilmes/crescimento & desenvolvimento , Complexo Burkholderia cepacia/crescimento & desenvolvimento , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Glicoproteínas/química , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Testes de Sensibilidade Microbiana , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Relação Estrutura-AtividadeRESUMO
The ability to maintain proper airway surface liquid (ASL) volume homeostasis is vital for mucus hydration and clearance, which are essential aspects of the mammalian lung's innate defense system. In cystic fibrosis (CF), one of the most common life-threatening genetic disorders, ASL dehydration leads to mucus accumulation and chronic infection. In normal airways, the secreted protein short palate lung and nasal epithelial clone 1 (SPLUNC1) effectively inhibits epithelial Na(+) channel (ENaC)-dependent Na(+) absorption and preserves ASL volume. In CF airways, it has been hypothesized that increased ENaC-dependent Na(+) absorption contributes to ASL depletion, and hence increased disease. However, this theory is controversial, and the mechanism for abnormal ENaC regulation in CF airways has remained elusive. Here, we show that SPLUNC1 is a pH-sensitive regulator of ENaC and is unable to inhibit ENaC in the acidic CF airway environment. Alkalinization of CF airway cultures prevented CF ASL hyperabsorption, and this effect was abolished when SPLUNC1 was stably knocked down. Accordingly, we resolved the crystal structure of SPLUNC1 to 2.8 Å. Notably, this structure revealed two pH-sensitive salt bridges that, when removed, rendered SPLUNC1 pH-insensitive and able to regulate ASL volume in acidic ASL. Thus, we conclude that ENaC hyperactivity is secondary to reduced CF ASL pH. Together, these data provide molecular insights into the mucosal dehydration associated with a range of pulmonary diseases, including CF, and suggest that future therapy be directed toward alkalinizing the pH of CF airways.
Assuntos
Fibrose Cística/patologia , Desidratação/metabolismo , Canais Epiteliais de Sódio/metabolismo , Glicoproteínas/química , Modelos Moleculares , Muco/química , Fosfoproteínas/química , Mucosa Respiratória/química , Adulto , Análise de Variância , Células Cultivadas , Cristalização , Fibrose Cística/complicações , Desidratação/etiologia , Desidratação/patologia , Técnicas de Silenciamento de Genes , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Concentração de Íons de Hidrogênio , North Carolina , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologiaRESUMO
Computational design of novel protein-protein interfaces is a test of our understanding of protein interactions and has the potential to allow modification of cellular physiology. Methods for designing high-affinity interactions that adopt a predetermined binding mode have proved elusive, suggesting the need for new strategies that simplify the design process. A solvent-exposed backbone on a ß-strand is thought of as "sticky" and ß-strand pairing stabilizes many naturally occurring protein complexes. Here, we computationally redesign a monomeric protein to form a symmetric homodimer by pairing exposed ß-strands to form an intermolecular ß-sheet. A crystal structure of the designed complex closely matches the computational model (rmsd = 1.0 Å). This work demonstrates that ß-strand pairing can be used to computationally design new interactions with high accuracy.
Assuntos
Proteínas/química , Cristalografia por Raios X/métodos , DNA/química , Dimerização , Escherichia coli/metabolismo , Luz , Conformação Molecular , Conformação Proteica , Engenharia de Proteínas/métodos , Estrutura Secundária de Proteína , Espalhamento de Radiação , Software , Solventes , Propriedades de Superfície , Termodinâmica , UltracentrifugaçãoRESUMO
Human immunodeficiency virus type 1 typically requires a high density of CD4 for efficient entry as a mechanism to target CD4+ T cells (T-tropic), with CCR5 being used most often as the coreceptor. When target T cells are limiting, the virus can evolve to infect cells with a low density of CD4 such as macrophages (M-tropic). The entry phenotype is known to be encoded in the viral Env protein on the surface of the virus particle. Using data showing a dose response for infectivity based on CD4 surface density, we built a model consistent with T-tropic viruses requiring multiple CD4 molecules to mediate infection, whereas M-tropic viruses can infect cells using a single CD4 receptor molecule interaction. We also found that T-tropic viruses bound to the surface of cells with a low density of CD4 are released more slowly than M-tropic viruses which we modeled to be due to multiple interactions of the T-tropic virus with multiple CD4 molecules to allow the initial stable binding. Finally, we found that some M-tropic Env proteins, as the gp120 subunit, possess an enhanced affinity for CD4 compared with their T-tropic pair, indicating that the evolution of macrophage tropism can be reflected both in the closed Env trimer conformation on the virion surface and, in some cases, also in the open confirmation of gp120 Env. Collectively, these studies reveal differences in the stoichiometry of interaction of T-tropic and M-tropic viruses with CD4 and start to identify the basis of binding differences at the biochemical level. IMPORTANCE: Human immunodeficiency virus type 1 normally targets CD4+ T cells for viral replication. When T cells are limiting, the virus can evolve to infect myeloid cells. The evolutionary step involves a change from requiring a high surface density of CD4 for entry to being able to infect cells with a low density of CD4, as is found on myeloid lineage cells such as macrophage and microglia. Viruses able to infect macrophages efficiently are most often found in the CNS late in the disease course, and such viruses may contribute to neurocognitive impairment. Here, we examine the CD4 binding properties of the viral Env protein to explore these two different entry phenotypes.
Assuntos
HIV-1 , Humanos , Antígenos CD4/metabolismo , Linfócitos T CD4-Positivos , Produtos do Gene env/metabolismo , HIV-1/fisiologia , Macrófagos/metabolismo , Receptores CCR5/metabolismo , Proteínas do Envelope Viral/metabolismo , Produtos do Gene env do Vírus da Imunodeficiência HumanaRESUMO
The short cytoplasmic tails of the α- and ß-chains of integrin adhesion receptors regulate integrin activation and cell signaling. Significantly less is known about proteins that bind to α-integrin cytoplasmic tails (CTs) as opposed to ß-CTs to regulate integrins. Calcium and integrin binding protein 1 (CIB1) was previously identified as an αIIb binding partner that inhibits agonist-induced activation of the platelet-specific integrin, αIIbß3. A sequence alignment of all α-integrin CTs revealed that key residues in the CIB1 binding site of αIIb are well-conserved, and was used to delineate a consensus binding site (I/L-x-x-x-L/M-W/Y-K-x-G-F-F). Because the CIB1 binding site of αIIb is conserved in all α-integrins and CIB1 expression is ubiquitous, we asked if CIB1 could interact with other α-integrin CTs. We predicted that multiple α-integrin CTs were capable of binding to the same hydrophobic binding pocket on CIB1 with docking models generated by all-atom replica exchange discrete molecular dynamics. After demonstrating novel in vivo interactions between CIB1 and other whole integrin complexes with co-immunoprecipitations, we validated the modeled predictions with solid-phase competitive binding assays, which showed that other α-integrin CTs compete with the αIIb CT for binding to CIB1 in vitro. Isothermal titration calorimetry measurements indicated that this binding is driven by hydrophobic interactions and depends on residues in the CIB1 consensus binding site. These new mechanistic details of CIB1-integrin binding imply that CIB1 could bind to all integrin complexes and act as a broad regulator of integrin function.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Cadeias alfa de Integrinas/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Ligação ao Cálcio/genética , Interações Hidrofóbicas e Hidrofílicas , Simulação de Dinâmica Molecular , Glicoproteína IIb da Membrana de Plaquetas/metabolismo , Ligação Proteica , Alinhamento de SequênciaRESUMO
The nuclear pore complex gates nucleocytoplasmic transport through a massive, eight-fold symmetric channel capped by a nucleoplasmic basket and structurally unique, cytoplasmic fibrils whose tentacles bind and regulate asymmetric traffic. The conserved Nup82 complex, composed of Nsp1, Nup82, and Nup159, forms the unique cytoplasmic fibrils that regulate mRNA nuclear export. Although the nuclear pore complex plays a fundamental, conserved role in nuclear trafficking, structural information about the cytoplasmic fibrils is limited. Here, we investigate the structural and biochemical interactions between Saccharomyces cerevisiae Nup159 and the nucleoporin, Dyn2. We find that Dyn2 is predominantly a homodimer and binds arrayed sites on Nup159, promoting the Nup159 parallel homodimerization. We present the first structure of Dyn2, determined at 1.85 Å resolution, complexed with a Nup159 target peptide. Dyn2 resembles homologous metazoan dynein light chains, forming homodimeric composite substrate binding sites that engage two independent 10-residue target motifs, imparting a ß-strand structure to each peptide via antiparallel extension of the Dyn2 core ß-sandwich. Dyn2 recognizes a highly conserved QT motif while allowing sequence plasticity in the flanking residues of the peptide. Isothermal titration calorimetric analysis of the comparative binding of Dyn2 to two Nup159 target sites shows similar affinities (18 and 13 µM), but divergent thermal binding modes. Dyn2 homodimers are arrayed in the crystal lattice, likely mimicking the arrayed architecture of Dyn2 on the Nup159 multivalent binding sites. Crystallographic interdimer interactions potentially reflect a cooperative basis for Dyn2-Nup159 complex formation. Our data highlight the determinants that mediate oligomerization of the Nup82 complex and promote a directed, elongated cytoplasmic fibril architecture.
Assuntos
Aciltransferases/química , Dineínas/química , Complexos Multiproteicos/química , Complexo de Proteínas Formadoras de Poros Nucleares/química , Proteínas de Saccharomyces cerevisiae/química , Aciltransferases/genética , Aciltransferases/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/genética , Ligação Competitiva , Calorimetria/métodos , Cristalografia por Raios X , Dineínas/genética , Dineínas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Protein lysine methyltransferases G9a and GLP modulate the transcriptional repression of a variety of genes via dimethylation of Lys9 on histone H3 (H3K9me2) as well as dimethylation of non-histone targets. Here we report the discovery of UNC0638, an inhibitor of G9a and GLP with excellent potency and selectivity over a wide range of epigenetic and non-epigenetic targets. UNC0638 treatment of a variety of cell lines resulted in lower global H3K9me2 levels, equivalent to levels observed for small hairpin RNA knockdown of G9a and GLP with the functional potency of UNC0638 being well separated from its toxicity. UNC0638 markedly reduced the clonogenicity of MCF7 cells, reduced the abundance of H3K9me2 marks at promoters of known G9a-regulated endogenous genes and disproportionately affected several genomic loci encoding microRNAs. In mouse embryonic stem cells, UNC0638 reactivated G9a-silenced genes and a retroviral reporter gene in a concentration-dependent manner without promoting differentiation.
Assuntos
Inibidores Enzimáticos/farmacologia , Histona-Lisina N-Metiltransferase/metabolismo , Quinazolinas/farmacologia , Animais , Linhagem Celular , Inativação Gênica , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/genética , Humanos , Camundongos , Estrutura MolecularRESUMO
Immune checkpoint inhibitors that bind to the cell surface receptor PD-L1 are effective anti-cancer agents but suffer from immune-related adverse events as PD-L1 is expressed on both healthy and cancer cells. To mitigate toxicity, researchers are testing prodrugs that have low affinity for checkpoint targets until activated with proteases enriched in the tumor microenvironment. Here, we engineer a prodrug form of a PD-L1 inhibitor. The inhibitor is a soluble PD-1 mimetic that was previously engineered to have high affinity for PD-L1. In the basal state, the binding surface of the PD-1 mimetic is masked by fusing it to a soluble variant of its natural ligand, PD-L1. Proteolytic cleavage of the linker that connects the mask to the inhibitor activates the molecule. To optimize the mask so that it effectively blocks binding to PD-L1 but releases upon cleavage, we tested a set of mutants with varied affinity for the inhibitor. The top-performing mask reduces the affinity of the prodrug for PD-L1 120-fold, and binding is nearly fully recovered upon cleavage. In a cell-based assay measuring inhibition of the PD-1:PD-L1 interaction on the surface of cells, the IC50s of the masked inhibitors were up to 40-fold higher than their protease-treated counterparts. The changes in activity we observe upon protease treatment are comparable to systems currently tested in the clinic and provide evidence that natural binding partners are an excellent starting point for creating a prodrug.
Assuntos
Inibidores de Checkpoint Imunológico , Pró-Fármacos , Antígeno B7-H1/metabolismo , Peptídeo Hidrolases , Receptor de Morte Celular Programada 1/metabolismoRESUMO
Many protein therapeutics are competitive inhibitors that function by binding to endogenous proteins and preventing them from interacting with native partners. One effective strategy for engineering competitive inhibitors is to graft structural motifs from a native partner into a host protein. Here, we develop and experimentally test a computational protocol for embedding binding motifs in de novo designed proteins. The protocol uses an "inside-out" approach: Starting with a structural model of the binding motif docked against the target protein, the de novo protein is built by growing new structural elements off the termini of the binding motif. During backbone assembly, a score function favors backbones that introduce new tertiary contacts within the designed protein and do not introduce clashes with the target binding partner. Final sequences are designed and optimized using the molecular modeling program Rosetta. To test our protocol, we designed small helical proteins to inhibit the interaction between Gα q and its effector PLC-ß isozymes. Several of the designed proteins remain folded above 90°C and bind to Gα q with equilibrium dissociation constants tighter than 80 nM. In cellular assays with oncogenic variants of Gα q , the designed proteins inhibit activation of PLC-ß isozymes and Dbl-family RhoGEFs. Our results demonstrate that computational protein design, in combination with motif grafting, can be used to directly generate potent inhibitors without further optimization via high throughput screening or selection. statement for broader audience: Engineered proteins that bind to specific target proteins are useful as research reagents, diagnostics, and therapeutics. We used computational protein design to engineer de novo proteins that bind and competitively inhibit the G protein, Gα q , which is an oncogene for uveal melanomas. This computational method is a general approach that should be useful for designing competitive inhibitors against other proteins of interest.
RESUMO
Many protein therapeutics are competitive inhibitors that function by binding to endogenous proteins and preventing them from interacting with native partners. One effective strategy for engineering competitive inhibitors is to graft structural motifs from a native partner into a host protein. Here, we develop and experimentally test a computational protocol for embedding binding motifs in de novo designed proteins. The protocol uses an "inside-out" approach: Starting with a structural model of the binding motif docked against the target protein, the de novo protein is built by growing new structural elements off the termini of the binding motif. During backbone assembly, a score function favors backbones that introduce new tertiary contacts within the designed protein and do not introduce clashes with the target binding partner. Final sequences are designed and optimized using the molecular modeling program Rosetta. To test our protocol, we designed small helical proteins to inhibit the interaction between Gαq and its effector PLC-ß isozymes. Several of the designed proteins remain folded above 90°C and bind to Gαq with equilibrium dissociation constants tighter than 80 nM. In cellular assays with oncogenic variants of Gαq , the designed proteins inhibit activation of PLC-ß isozymes and Dbl-family RhoGEFs. Our results demonstrate that computational protein design, in combination with motif grafting, can be used to directly generate potent inhibitors without further optimization via high throughput screening or selection.
Assuntos
Proteínas de Ligação ao GTP , Isoenzimas , Ligação Proteica , Modelos Moleculares , Engenharia de Proteínas/métodosRESUMO
Using the molecular modeling program Rosetta, we designed a de novo protein, called SEWN0.1, which binds the heterotrimeric G protein Gαq. The design is helical, well-folded, and primarily monomeric in solution at a concentration of 10 µM. However, when we solved the crystal structure of SEWN0.1 at 1.9 Å, we observed a dimer in a conformation incompatible with binding Gαq . Unintentionally, we had designed a protein that adopts alternate conformations depending on its oligomeric state. Recently, there has been tremendous progress in the field of protein structure prediction as new methods in artificial intelligence have been used to predict structures with high accuracy. We were curious if the structure prediction method AlphaFold could predict the structure of SEWN0.1 and if the prediction depended on oligomeric state. When AlphaFold was used to predict the structure of monomeric SEWN0.1, it produced a model that resembles the Rosetta design model and is compatible with binding Gαq , but when used to predict the structure of a dimer, it predicted a conformation that closely resembles the SEWN0.1 crystal structure. AlphaFold's ability to predict multiple conformations for a single protein sequence should be useful for engineering protein switches.
Assuntos
Inteligência Artificial , Proteínas , Sequência de Aminoácidos , Modelos Moleculares , Conformação Proteica , Proteínas/químicaRESUMO
A high number of leucocytes reside in the human endometrium and are distributed differentially during the menstrual cycle and pregnancy. During early pregnancy, decidual natural killer (dNK) cells are the most common type of natural killer (NK) cells in the uterus. The increase in the number of uterine NK (uNK) cells during the mid-secretory phase of the menstrual cycle, followed by further increase of dNK cells in early pregnancy, has heightened interest in their involvement during pregnancy. Extensive research has revealed various roles of dNK cells during pregnancy including the formation of new blood vessels, migration of trophoblasts, and immunological tolerance. The present review article is focused on the significance of NK cells during pregnancy and their role in pregnancy-related diseases. The article will provide an in-depth review of cellular and molecular interactions during pregnancy and related disorders, with NK cells playing a pivotal role. Moreover, this study will help researchers to understand the physiology of normal pregnancy and related complications with respect to NK cells, so that future research work can be designed to alleviate the complications.
Assuntos
Decídua/imunologia , Tolerância Imunológica , Células Matadoras Naturais/imunologia , Complicações na Gravidez/imunologia , Trofoblastos/imunologia , Feminino , Humanos , GravidezRESUMO
The heparin-binding hemagglutinin adhesin (HBHA) is a surface adhesin on the human pathogen Mycobacterium tuberculosis. Previously, it has been shown that HBHA exists as a dimer in solution. We investigated the detailed nature of this dimer using circular dichroism spectroscopy and analytical ultracentrifugation techniques. We demonstrate that the heparan sulfate (HS) binding region does not play a role in dimerization in solution, while the linker region between the predicted N-terminal coiled-coil and the C-terminal HS binding region does affect dimer stability. The majority of contacts responsible for dimerization, folding, and stability lie within the predicted coiled-coil region of HBHA, while the N-terminal helix preceding the coiled-coil appears to trigger the folding and dimerization of HBHA. Constructs lacking this initial helix or containing site-specific mutations produce nonhelical monomers in solution. Thus, we show that HBHA dimerization and folding are linked and that the N-terminal region of this cell surface adhesin triggers the formation of an HBHA coiled-coil dimer.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Proteínas de Membrana/metabolismo , Mycobacterium tuberculosis/metabolismo , Dobramento de Proteína , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Clonagem Molecular , Proteínas de Membrana/genética , Dados de Sequência Molecular , Conformação ProteicaRESUMO
In humans the mitochondrial inner membrane protein Oxa1L is involved in the biogenesis of membrane proteins and facilitates the insertion of both mitochondrial- and nuclear-encoded proteins from the mitochondrial matrix into the inner membrane. The C-terminal approximately 100-amino acid tail of Oxa1L (Oxa1L-CTT) binds to mitochondrial ribosomes and plays a role in the co-translational insertion of mitochondria-synthesized proteins into the inner membrane. Contrary to suggestions made for yeast Oxa1p, our results indicate that the C-terminal tail of human Oxa1L does not form a coiled-coil helical structure in solution. The Oxa1L-CTT exists primarily as a monomer in solution but forms dimers and tetramers at high salt concentrations. The binding of Oxa1L-CTT to mitochondrial ribosomes is an enthalpy-driven process with a K(d) of 0.3-0.8 microM and a stoichiometry of 2. Oxa1L-CTT cross-links to mammalian mitochondrial homologs of the bacterial ribosomal proteins L13, L20, and L28 and to mammalian mitochondrial specific ribosomal proteins MRPL48, MRPL49, and MRPL51. Oxa1L-CTT does not cross-link to proteins decorating the conventional exit tunnel of the bacterial large ribosomal subunit (L22, L23, L24, and L29).