Structural and functional evaluation of de novo-designed, two-component nanoparticle carriers for HIV Env trimer immunogens

AbstractTwo-component, self-assembling nanoparticles represent a versatile platform for multivalent presentation of viral antigens. Nanoparticles of different sizes and geometries can be designed and combined with appropriate antigens to fit the requirements of different immunization strategies. Here, we describe detailed antigenic, structural, and functional characterization of computationally designed tetrahedral, octahedral, and icosahedral nanoparticle immunogens displaying trimeric HIV envelope glycoprotein (Env) ectodomains. Env trimers, based on subtype A (BG505) or consensus group M (ConM) sequences and engineered with SOSIP stabilizing mutations, were fused to the underlying trimeric building block of each nanoparticle. Initial screening yielded one icosahedral and two tetrahedral nanoparticle candidates, capable of presenting twenty or four copies of the Env trimer. A number of analyses, including detailed structural characterization by cryo-EM, demonstrated that the nanoparticle immunogens possessed the intended structural and antigenic properties. Comparing the humoral responses elicited by ConM-SOSIP trimers presented on a two-component tetrahedral nanoparticle to the corresponding soluble protein revealed that multivalent presentation increased the proportion of the overall antibody response directed against autologous neutralizing Ab epitopes present on the ConM-SOSIP trimers.Author SummaryProtein constructs based on soluble ectodomains of HIV glycoprotein (Env) trimers are the basis of many current HIV vaccine platforms. Multivalent antigen display is one strategy applied to improve the immunogenicity of different subunit vaccine candidates. Here, we describe and comprehensively evaluate a library of de novo designed, protein nanoparticles of different geometries for their ability to present trimeric Env antigens. We found three nanoparticle candidates that can stably incorporate model Env trimer on their surface while maintaining its structure and antigenicity. Immunogenicity of the designed nanoparticles is assessed in vitro and in vivo. In addition to introducing a novel set of reagents for multivalent display of Env trimers, this work provides both guiding principles and a detailed experimental roadmap for the generation, characterization, and optimization of Env-presenting, self-assembling nanoparticle immunogens.

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De novo design of self-assembling helical protein filaments

Science ◽

10.1126/science.aau3775 ◽

2018 ◽

Vol 362 (6415) ◽

pp. 705-709 ◽

Cited By ~ 48

Author(s):

Hao Shen ◽

Jorge A. Fallas ◽

Eric Lynch ◽

William Sheffler ◽

Bradley Parry ◽

...

Keyword(s):

De Novo ◽

Computational Design ◽

Building Blocks ◽

Repeat Units ◽

Self Assembling ◽

Wide Range ◽

Helical Protein ◽

Monomeric Proteins

We describe a general computational approach to designing self-assembling helical filaments from monomeric proteins and use this approach to design proteins that assemble into micrometer-scale filaments with a wide range of geometries in vivo and in vitro. Cryo–electron microscopy structures of six designs are close to the computational design models. The filament building blocks are idealized repeat proteins, and thus the diameter of the filaments can be systematically tuned by varying the number of repeat units. The assembly and disassembly of the filaments can be controlled by engineered anchor and capping units built from monomers lacking one of the interaction surfaces. The ability to generate dynamic, highly ordered structures that span micrometers from protein monomers opens up possibilities for the fabrication of new multiscale metamaterials.

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New Twists and Turns in Bacterial Locomotion and Signal Transduction

Journal of Bacteriology ◽

10.1128/jb.00439-19 ◽

2019 ◽

Vol 201 (20) ◽

Cited By ~ 2

Author(s):

Kylie J. Watts ◽

Ady Vaknin ◽

Clay Fuqua ◽

Barbara I. Kazmierczak

Keyword(s):

Signal Transduction ◽

De Novo ◽

Electron Tomography ◽

Bacterial Cells ◽

Research Areas ◽

Flagellar Motors ◽

Two Component ◽

Two Component Signal Transduction

ABSTRACT Prokaryotic organisms occupy the most diverse set of environments and conditions on our planet. Their ability to sense and respond to a broad range of external cues remain key research areas in modern microbiology, central to behaviors that underlie beneficial and pathogenic interactions of bacteria with multicellular organisms and within complex ecosystems. Advances in our understanding of the one- and two-component signal transduction systems that underlie these sensing pathways have been driven by advances in imaging the behavior of many individual bacterial cells, as well as visualizing individual proteins and protein arrays within living cells. Cryo-electron tomography continues to provide new insights into the structure and function of chemosensory receptors and flagellar motors, while advances in protein labeling and tracking are applied to understand information flow between receptor and motor. Sophisticated microfluidics allow simultaneous analysis of the behavior of thousands of individual cells, increasing our understanding of how variance between individuals is generated, regulated, and employed to maximize fitness of a population. In vitro experiments have been complemented by the study of signal transduction and motility in complex in vivo models, allowing investigators to directly address the contribution of motility, chemotaxis, and aggregation/adhesion on virulence during infection. Finally, systems biology approaches have demonstrated previously uncharted areas of protein space in which novel two-component signal transduction pathways can be designed and constructed de novo. These exciting experimental advances were just some of the many novel findings presented at the 15th Bacterial Locomotion and Signal Transduction conference (BLAST XV) in January 2019.

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Aberrant Splicing Events Associated to CDH23 Noncanonical Splice Site Mutations in a Proband with Atypical Usher Syndrome 1

Genes ◽

10.3390/genes10100732 ◽

2019 ◽

Vol 10 (10) ◽

pp. 732 ◽

Cited By ~ 3

Author(s):

Rebeca Valero ◽

Marta de Castro-Miró ◽

Sofía Jiménez-Ochoa ◽

Juan José Rodríguez-Ezcurra ◽

Gemma Marfany ◽

...

Keyword(s):

Splice Site ◽

Usher Syndrome ◽

Functional Characterization ◽

Genetic Diagnosis ◽

Functional Evaluation ◽

Syndrome Type ◽

Intronic Variants ◽

Usher Syndrome Type

Aims: The aim of this study was the genetic diagnosis by next generation sequencing (NGS) of a patient diagnosed with Usher syndrome type 2 and the functional evaluation of the identified genetic variants to establish a phenotype–genotype correlation. Methods: Whole exome sequencing (WES) analysis identified two heterozygous intronic variants in CDH23, a gene responsible of Usher syndrome type 1. Evaluation of the putative splicing effects was performed in vivo, in whole blood samples, and in vitro, by transfection of midigene constructs in HEK293T cells. Results: Two intronic variants were identified in intron 45 of CDH23—one novel, c.6050-15G>A, and the other, c.6050-9G>A, already reported as a noncanonical splice site (NCSS) mutation—with partial functional characterization. In vivo and in vitro analyses showed aberrant transcripts by the addition of 13 and 7 nucleotides to exon 46, respectively. Transcript degradation by nonsense mediated decay (NMD) in blood cells could only be prevented by cycloheximide treatment. Midigene constructs showed that the two variants contributed to exon skipping and generated aberrantly spliced transcripts. Conclusions: A combination of in vivo and in vitro assays provided a comprehensive view of the physiological effects of NCSS variants, which in this case led to a clinical reassignment of the proband as affected with atypical USH1 syndrome.

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Cell Biological and Functional Characterization of the Vaccinia Virus F10 Kinase: Implications for the Mechanism of Virion Morphogenesis

Journal of Virology ◽

10.1128/jvi.79.4.2171-2190.2005 ◽

2005 ◽

Vol 79 (4) ◽

pp. 2171-2190 ◽

Cited By ~ 41

Author(s):

Almira Punjabi ◽

Paula Traktman

Keyword(s):

Enzymatic Activity ◽

Vaccinia Virus ◽

Catalytic Domain ◽

De Novo ◽

Functional Characterization ◽

Site Formation ◽

Phenotypic Analysis ◽

Virion Morphogenesis

ABSTRACT The vaccinia virus F10 protein is one of two virally encoded protein kinases. A phenotypic analysis of infections involving a tetracycline-inducible recombinant (vΔiF10) indicated that F10 is involved in the early stages of virion morphogenesis, as previously reported for the mutants ts28 and ts15. The proteins encoded by ts28 and ts15 have primary defects in enzymatic activity and thermostability, respectively. Using a transient complementation assay, we demonstrated that the enzymatic activity of F10 is essential for its biological function and that both its enzymatic and biological functions depend upon N-terminal sequences that precede the catalytic domain. An execution point analysis indicated that in addition to its role at the onset of morphogenesis, F10 is also required at later stages, when membrane crescents surround virosomal contents and develop into immature virions. The F10 protein is phosphorylated in vivo, appears to be tightly associated with intracellular membranes, and can bind to specific phosphoinositides in vitro. When F10 is repressed or impaired, the phosphorylation of several cellular and viral proteins appears to increase in intensity, suggesting that F10 may normally intersect with cellular signaling cascades via the activation of a phosphatase or the inhibition of another kinase. These cascades may drive the F10-induced remodeling of membranes that accompanies virion biogenesis. Upon the release of ts28-infected cultures from a 40°C-induced block, a synchronous resumption of morphogenesis that culminates in the production of infectious virus can be observed. The pharmacological agents H89 and cerulenin, which are inhibitors of endoplasmic reticulum exit site formation and de novo lipid synthesis, respectively, block this recovery.

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Functional characterization of RELN missense mutations involved in recessive and dominant forms of Neuronal Migration Disorders

10.1101/2021.05.25.445586 ◽

2021 ◽

Author(s):

Martina Riva ◽

Sofia Ferreira ◽

Vera P. Medvedeva ◽

Frédéric Causeret ◽

Olivia J. Henry ◽

...

Keyword(s):

Neuronal Migration ◽

De Novo ◽

Functional Characterization ◽

Valuable Insight ◽

Embryonic Mouse ◽

Cortical Malformations ◽

Neuronal Migration Disorders ◽

Migration Disorders

RELN is a large secreted glycoprotein that acts at multiple steps of cerebral cortex development, including neuronal migration. Only recessive mutations of the Reelin gene (RELN) have been associated with human cortical malformations and none has been functionally characterized. We identified novel missense RELN mutations in both compound and de novo heterozygous patients exhibiting an array of neuronal migration disorders (NMDs) as diverse as pachygyria, polymicrogyria and heterotopia. Most mutations caused defective RELN secretion in vitro and, when ectopically expressed in the embryonic mouse cortex, affected neuronal aggregation and/or migration in vivo. We determined the de novo heterozygous mutations acted as dominant negative and demonstrated that RELN mutations mediate not only recessive, but also dominant NMDs. This work assesses for the first time the pathogenicity of RELN mutations showing a strong genotype-phenotype correlation. In particular, the behavior of the mutant proteins in vitro and in vivo predicts the severity of cortical malformations and provides valuable insight into the pathogenesis of these disorders.

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Functional identification of ygiP as a positive regulator of the ttdA-ttdB-ygjE operon

Microbiology ◽

10.1099/mic.0.28753-0 ◽

2006 ◽

Vol 152 (7) ◽

pp. 2129-2135 ◽

Cited By ~ 17

Author(s):

Taku Oshima ◽

Francis Biville

Keyword(s):

Functional Characterization ◽

Anaerobic Growth ◽

Functional Identification ◽

New Members ◽

E Coli ◽

Inner Membrane Protein ◽

Tartrate Dehydratase

Functional characterization of unknown genes is currently a major task in biology. The search for gene function involves a combination of various in silico, in vitro and in vivo approaches. Available knowledge from the study of more than 21 LysR-type regulators in Escherichia coli has facilitated the classification of new members of the family. From sequence similarities and its location on the E. coli chromosome, it is suggested that ygiP encodes a lysR regulator controlling the expression of a neighbouring operon; this operon encodes the two subunits of tartrate dehydratase (TtdA, TtdB) and YgiE, an integral inner-membrane protein possibly involved in tartrate uptake. Expression of tartrate dehydratase, which converts tartrate to oxaloacetate, is required for anaerobic growth on glycerol as carbon source in the presence of tartrate. Here, it has been demonstrated that disruption of ygiP, ttdA or ygjE abolishes tartrate-dependent anaerobic growth on glycerol. It has also been shown that tartrate-dependent induction of the ttdA-ttdB-ygjE operon requires a functional YgiP.

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Functional Characterization of the mazEF Toxin-Antitoxin System in the Pathogenic Bacterium Agrobacterium tumefaciens

Microorganisms ◽

10.3390/microorganisms9051107 ◽

2021 ◽

Vol 9 (5) ◽

pp. 1107

Author(s):

Wonho Choi ◽

Yoshihiro Yamaguchi ◽

Ji-Young Park ◽

Sang-Hyun Park ◽

Hyeok-Won Lee ◽

...

Keyword(s):

Agrobacterium Tumefaciens ◽

Physiological Function ◽

Functional Characterization ◽

Site Directed Mutagenesis ◽

In Vitro Assays ◽

Cell Growth Inhibition ◽

The Right

Agrobacterium tumefaciens is a pathogen of various plants which transfers its own DNA (T-DNA) to the host plants. It is used for producing genetically modified plants with this ability. To control T-DNA transfer to the right place, toxin-antitoxin (TA) systems of A. tumefaciens were used to control the target site of transfer without any unintentional targeting. Here, we describe a toxin-antitoxin system, Atu0939 (mazE-at) and Atu0940 (mazF-at), in the chromosome of Agrobacterium tumefaciens. The toxin in the TA system has 33.3% identity and 45.5% similarity with MazF in Escherichia coli. The expression of MazF-at caused cell growth inhibition, while cells with MazF-at co-expressed with MazE-at grew normally. In vivo and in vitro assays revealed that MazF-at inhibited protein synthesis by decreasing the cellular mRNA stability. Moreover, the catalytic residue of MazF-at was determined to be the 24th glutamic acid using site-directed mutagenesis. From the results, we concluded that MazF-at is a type II toxin-antitoxin system and a ribosome-independent endoribonuclease. Here, we characterized a TA system in A. tumefaciens whose understanding might help to find its physiological function and to develop further applications.

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1455. Potential Mechanisms of Cefiderocol MIC Increase in Enterobacterales in In Vitro Resistance Acquisition Studies

Open Forum Infectious Diseases ◽

10.1093/ofid/ofaa439.1636 ◽

2020 ◽

Vol 7 (Supplement_1) ◽

pp. S730-S730

Author(s):

Yoshinori Yamano ◽

Rio Nakamura ◽

Miki Takemura ◽

Roger Echols

Keyword(s):

Iron Transport ◽

Resistance Mechanisms ◽

Altered Expression ◽

Carbapenem Resistant ◽

Mueller Hinton ◽

Two Component ◽

Mueller Hinton Agar ◽

Related Proteins

Abstract Background Cefiderocol (CFDC) is a novel siderophore, iron-chelating cephalosporin, which is transported into bacteria via iron transporters. CFDC has potent in vitro and in vivo activity against all aerobic Gram-negative bacteria, including carbapenem-resistant strains. To date, clinical isolates with cefiderocol MIC >4 µg/mL have been found infrequently, in which the presence of a few β-lactamases or altered iron transport was found. We investigated potential new mechanisms causing CFDC MIC increases in non-clinical studies. Methods The mutation positions were determined by whole genome sequencing using four K. pneumoniae mutants including two KPC producers and one NDM producer that had shown CFDC MIC increases in previous in vitro resistance-acquisition studies. The mutant strains were obtained at the frequency of 10-7 to < 10-8 by spreading bacteria on standard Mueller‒Hinton agar medium containing CFDC at concentrations of 10× MIC, with or without apo-transferrin (20 μg/mL). CFDC MIC was determined by broth microdilution using iron-depleted cation-adjusted Mueller-Hinton broth based on Clinical and Laboratory Standards Institute guidelines. The emergence of MIC increase mutants was also assessed by in vitro chemostat models under humanized plasma pharmacokinetic exposures of CFDC. Results The possible resistance mechanisms were investigated. Mutation of baeS or envZ, sensors of two-component regulation systems, were found in three or two mutants among the tested four isolates, respectively, and caused the MIC to increase by 4–32-fold. The altered expression level of specific genes by the baeS or envZ mutation could affect CFDC susceptibility, but the specific genes have not been identified. In addition, the mutation of exbD, an accessory protein related to iron transport, was identified in one case and caused the MIC to increase by >8-fold. In vitro chemostat studies using two isolates (one NDM producer and one KPC producer) showed no resistance acquisition during 24-hour exposure. Table. Overview of mutation emergence in five isolates of K. pneumoniae Conclusion The mutation of two-component regulation systems (BaeSR and OmpR/EnvZ) and iron transport-related proteins were shown to be possible mechanisms causing CFDC MIC increases, but these mutants did not appear under human exposures. Disclosures Yoshinori Yamano, PhD, Shionogi & Co., Ltd. (Employee) Rio Nakamura, BSc, Shionogi & Co., Ltd. (Employee) Miki Takemura, MSc, Shionogi & Co., Ltd. (Employee) Roger Echols, MD, Shionogi Inc. (Consultant)

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Computational study for suppression of CD25/IL-2 interaction

Biological Chemistry ◽

10.1515/hsz-2020-0326 ◽

2020 ◽

Vol 0 (0) ◽

Author(s):

Moein Dehbashi ◽

Zohreh Hojati ◽

Majid Motovali-bashi ◽

Mazdak Ganjalikhani-Hakemi ◽

Akihiro Shimosaka ◽

...

Keyword(s):

Small Molecules ◽

Cancer Progression ◽

Immune Escape ◽

De Novo ◽

Computational Study ◽

Treg Cells ◽

Homology Search ◽

Small Interfering Rnas

AbstractCancer recurrence presents a huge challenge in cancer patient management. Immune escape is a key mechanism of cancer progression and metastatic dissemination. CD25 is expressed in regulatory T (Treg) cells including tumor-infiltrating Treg cells (TI-Tregs). These cells specially activate and reinforce immune escape mechanism of cancers. The suppression of CD25/IL-2 interaction would be useful against Treg cells activation and ultimately immune escape of cancer. Here, software, web servers and databases were used, at which in silico designed small interfering RNAs (siRNAs), de novo designed peptides and virtual screened small molecules against CD25 were introduced for the prospect of eliminating cancer immune escape and obtaining successful treatment. We obtained siRNAs with low off-target effects. Further, small molecules based on the binding homology search in ligand and receptor similarity were introduced. Finally, the critical amino acids on CD25 were targeted by a de novo designed peptide with disulfide bond. Hence we introduced computational-based antagonists to lay a foundation for further in vitro and in vivo studies.

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EXTH-51. ANTI-INVASIVE EFFICACY AND SURVIVAL BENEFIT OF THE YAP-TEAD INHIBITOR VERTEPORFIN IN PRECLINICAL GLIOBLASTOMA MODELS

Neuro-Oncology ◽

10.1093/neuonc/noaa215.405 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii98-ii98

Author(s):

Anne Marie Barrette ◽

Alexandros Bouras ◽

German Nudelman ◽

Zarmeen Mussa ◽

Elena Zaslavsky ◽

...

Keyword(s):

Survival Benefit ◽

De Novo ◽

Epithelial Mesenchymal Transition ◽

Intraperitoneal Administration ◽

Standard Of Care ◽

Tumor Burden ◽

Mesenchymal Transition ◽

Protein Levels

Abstract Glioblastoma (GBM) remains an incurable disease, in large part due to its malignant infiltrative spread, and current clinical therapy fails to target the invasive nature of tumor cells in disease progression and recurrence. Here, we use the YAP-TEAD inhibitor Verteporfin to target a convergence point for regulating tumor invasion/metastasis and establish the robust anti-invasive therapeutic efficacy of this FDA-approved drug and its survival benefit across several preclinical glioma models. Using patient-derived GBM cells and orthotopic xenograft models (PDX), we show that Verteporfin treatment disrupts YAP/TAZ-TEAD activity and processes related to cell adhesion, migration and epithelial-mesenchymal transition. In-vitro, Verteporfin impairs tumor migration, invasion and motility dynamics. In-vivo, intraperitoneal administration of Verteporfin in mice with orthotopic PDX tumors shows consistent drug accumulation within the brain and decreased infiltrative tumor burden, across three independent experiments. Interestingly, PDX tumors with impaired invasion after Verteporfin treatment downregulate CDH2 and ITGB1 adhesion protein levels within the tumor microenvironment. Finally, Verteporfin treatment confers survival benefit in two independent PDX models: as monotherapy in de-novo GBM and in combination with standard-of-care chemoradiation in recurrent GBM. These findings indicate potential therapeutic value of this FDA-approved drug if repurposed for GBM patients.

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