Design of mimotopes of a conserved epitope in dengue and Zika viruses for the obtention of broadly neutralizing antibodies

Zika and dengue viruses are members of the Flavivirus genus that share many structural and pathological characteristics. They cause mild fever, rash and general body pain but can cause severe reactions, as hemorrhages (dengue virus), congenital syndrome (Zika virus), or even death. After an infection, virus-specific antibodies are generated by the immune system; however, because of the structural similarity between these viruses, some antibodies can cross-react with different members of the flavivirus family. After a secondary infection, the cross-reactive antibodies can lead to the more severe forms of the disease, through a mechanism named antibody-dependent enhancement of infection (ADE). Recently, some broadly neutralizing antibodies (antibodies that neutralize both, dengue and Zika, viruses), have been isolated and it has been demonstrated that they do not induce ADE. These antibodies are directed to a discontinuous quaternary epitope named the Envelope Dimer Epitope (EDE)1, located in the envelope (E) protein of both viruses. To obtain EDE, it is necessary to express the complete E protein, which contains other epitopes that induce ADE. This study aims to generate peptides that emulate the EDE epitope structure (mimotopes) without inducing ADE, and study its capacity to elicit broadly neutralizing antibodies against dengue and Zika viruses, to obtain a vaccine candidate for both viruses. Please click Download on the upper right corner to see the full abstract

Design of mimotopes of a conserved epitope in dengue and Zika viruses for the obtention of broadly neutralizing antibodies

Zika and dengue viruses are members of the Flavivirus genus that share many structural and pathological characteristics. They cause mild fever, rash and general body pain but can cause severe reactions, as hemorrhages (dengue virus), congenital syndrome (Zika virus), or even death. After an infection, virus-specific antibodies are generated by the immune system; however, because of the structural similarity between these viruses, some antibodies can cross-react with different members of the flavivirus family. After a secondary infection, the cross-reactive antibodies can lead to the more severe forms of the disease, through a mechanism named antibody-dependent enhancement of infection (ADE). Recently, some broadly neutralizing antibodies (antibodies that neutralize both, dengue and Zika, viruses), have been isolated and it has been demonstrated that they do not induce ADE. These antibodies are directed to a discontinuous quaternary epitope named the Envelope Dimer Epitope (EDE)1, located in the envelope (E) protein of both viruses. To obtain EDE, it is necessary to express the complete E protein, which contains other epitopes that induce ADE. This study aims to generate peptides that emulate the EDE epitope structure (mimotopes) without inducing ADE, and study its capacity to elicit broadly neutralizing antibodies against dengue and Zika viruses, to obtain a vaccine candidate for both viruses. Please click Download on the upper right corner to see the full abstract

Design of a vaccine against dengue and Zika viruses based on a mimotope of the envelope dimer epitope

Zika and dengue viruses are members of the Flavivirus genus that cause mild fever, rash and general body pain; but can cause severe reactions, as hemorrhages (dengue virus), congenital syndrome (Zika virus), or even death. Because of the structural similarity between these viruses, some antibodies generated after an infection can cross-react with different members of the flavivirus family. After a secondary infection, the cross-reactive antibodies can lead to more severe forms of the disease, through a mechanism named antibody-dependent enhancement of infection (ADE). Broadly neutralizing antibodies are antibodies that neutralize both, dengue and Zika viruses; and it has been demonstrated that they do not induce ADE. These antibodies are directed to a discontinuous quaternary epitope named the Envelope Dimer Epitope (EDE)1, located in the envelope (E) protein. To obtain the EDE, it is necessary to express the complete E protein, which contains other epitopes that induce ADE. This study aims to generate a peptide that emulates the EDE epitope structure (mimotope) in order to be used as a dual vaccine against dengue and Zika viruses; without causing ADE. Please click Download on the upper right corner to see the full abstract

G-Quadruplex (G4) Motifs in the Maize (Zea mays L.) Genome Are Enriched at Specific Locations in Thousands of Genes Coupled to Energy Status, Hypoxia, Low Sugar, and Nutrient Deprivation

The G-quadruplex (G4) elements comprise a class of nucleic acid structures formed by stacking of guanine base quartets in a quadruple helix. This G4 DNA can form within or across single-stranded DNA molecules and is mutually exclusive with duplex B-form DNA. The reversibility and structural diversity of G4s make them highly versatile genetic structures, as demonstrated by their roles in various functions including telomere metabolism, genome maintenance, immunoglobulin gene diversification, transcription, and translation. Sequence motifs capable of forming G4 DNA are typically located in telomere repeat DNA and other non-telomeric genomic loci. To investigate their potential roles in a large-genome model plant species, we computationally identified 149,988 non-telomeric G4 motifs in maize (Zea mays L., B73 AGPv2), 29% of which were in non-repetitive genomic regions. G4 motif hotspots exhibited non-random enrichment in genes at two locations on the antisense strand, one in the 5′ UTR and the other at the 5′ end of the first intron. Several genic G4 motifs were shown to adopt sequence-specific and potassium-dependent G4 DNA structures in vitro. The G4 motifs were prevalent in key regulatory genes associated with hypoxia (group VII ERFs), oxidative stress (DJ-1/GATase1), and energy status (AMPK/SnRK) pathways. They also showed statistical enrichment for genes in metabolic pathways that function in glycolysis, sugar degradation, inositol metabolism, and base excision repair. Collectively, the maize G4 motifs may represent conditional regulatory elements that can aid in energy status gene responses. Such a network of elements could provide a mechanistic basis for linking energy status signals to gene regulation in maize, a model genetic system and major world crop species for feed, food, and fuel

Bulged and Canonical G-Quadruplex Conformations Determine NDPK Binding Specificity

Guanine-rich DNA strands can adopt tertiary structures known as G-quadruplexes (G4s) that form when Hoogsteen base-paired guanines assemble as planar stacks, stabilized by a central cation like K+. In this study, we investigated the conformational heterogeneity of a G-rich sequence from the 5′ untranslated region of the Zea mays hexokinase4 gene. This sequence adopted an extensively polymorphic G-quadruplex, including non-canonical bulged G-quadruplex folds that co-existed in solution. The nature of this polymorphism depended, in part, on the incorporation of different sets of adjacent guanines into a quadruplex core, which permitted the formation of the different conformations. Additionally, we showed that the maize homolog of the human nucleoside diphosphate kinase (NDPK) NM23-H2 protein—ZmNDPK1—specifically recognizes and promotes formation of a subset of these conformations. Heteromorphic G-quadruplexes play a role in microorganisms’ ability to evade the host immune system, so we also discuss how the underlying properties that determine heterogeneity of this sequence could apply to microorganism G4s

The Maize (<i>Zea mays</i> L.) <i>Nucleoside Diphosphate Kinase1 (ZmNDPK1)</i> Gene Encodes a Human NM23-H2 Homologue That Binds and Stabilizes G‑Quadruplex DNA

Noncanonical forms of DNA like the guanine quadruplex (G4) play important roles in regulating transcription and translation through interactions with their protein partners. Although potential G4 elements have been identified in or near genes from species diverse as bacteria, mammals, and plants, little is known about how they might function as <i>cis</i>-regulatory elements or as binding sites for <i>trans</i>-acting protein partners. In fact, until now no G4 binding partners have been identified in the plant kingdom. Here, we report on the cloning and characterization of the first plant-kingdom gene known to encode a G4-binding protein, maize (<i>Zea mays</i> L.) <i>nucleoside diphosphate kinase1 (ZmNDPK1)</i>. Structural characterization by X-ray crystallography reveals that it is a homohexamer, akin to other known NDPKs like the human homologue NM23-H2. Further probing into the G4-binding properties of both NDPK homologues suggests that ZmNDPK1 possesses properties distinct from that of NM23-H2, which is known to interact with a G-rich sequence element upstream of the <i>c-myc</i> gene and, in doing so, modulate its expression. Indeed, ZmNDPK1 binds the folded G4 with low nanomolar affinity but corresponding unfolded G-rich DNA more weakly, whereas NM23-H2 binds both folded and unfolded G4 with low nanomolar affinities; nonetheless, both homologues appear to stabilize folded DNAs whether they were prefolded or not. We also demonstrate that the G4-binding activity of ZmNDPK1 is independent of nucleotide binding and kinase activity, suggesting that the G4-binding region and the enzyme active sites are separate. Together, these findings establish a broad evolutionary conservation of some NDPKs as G4-DNA binding enzymes, but with potentially distinct biochemical properties that may reflect divergent evolution or species-specific deployment of these elements in gene regulatory processes

Label-free visual proteomics: Coupling MS- and EM-based approaches in structural biology

Combining diverse experimental structural and interactomic methods allows for the construction of comprehensible molecular encyclopedias of biological systems. Typically, this involves merging several independent approaches that provide complementary structural and functional information from multiple perspectives and at different resolution ranges. A particularly potent combination lies in coupling structural information from cryoelectron microscopy or tomography (cryo-EM or cryo-ET) with interactomic and structural information from mass spectrometry (MS)-based structural proteomics. Cryo-EM/ET allows for sub-nanometer visualization of biological specimens in purified and near-native states, while MS provides bioanalytical information for proteins and protein complexes without introducing additional labels. Here we highlight recent achievements in protein structure and interactome determination using cryo-EM/ET that benefit from additional MS analysis. We also give our perspective on how combining cryo-EM/ET and MS will continue bridging gaps between molecular and cellular studies by capturing and describing 3D snapshots of proteomes and interactomes

The structure of natively iodinated bovine thyroglobulin

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/171011/1/ayd2jb5028.pd

G-Quadruplex (G4) Motifs in the Maize (Zea mays L.) Genome Are Enriched at Specific Locations in Thousands of Genes Coupled to Energy Status, Hypoxia, Low Sugar, and Nutrient Deprivation

The G-quadruplex (G4) elements comprise a class of nucleic acid structures formed by stacking of guanine base quartets in a quadruple helix. This G4 DNA can form within or across single-stranded DNA molecules and is mutually exclusive with duplex B-form DNA. The reversibility and structural diversity of G4s make them highly versatile genetic structures, as demonstrated by their roles in various functions including telomere metabolism, genome maintenance, immunoglobulin gene diversification, transcription, and translation. Sequence motifs capable of forming G4 DNA are typically located in telomere repeat DNA and other non-telomeric genomic loci. To investigate their potential roles in a large-genome model plant species, we computationally identified 149,988 non-telomeric G4 motifs in maize (Zea mays L., B73 AGPv2), 29% of which were in non-repetitive genomic regions. G4 motif hotspots exhibited non-random enrichment in genes at two locations on the antisense strand, one in the 5′ UTR and the other at the 5′ end of the first intron. Several genic G4 motifs were shown to adopt sequence-specific and potassium-dependent G4 DNA structures in vitro. The G4 motifs were prevalent in key regulatory genes associated with hypoxia (group VII ERFs), oxidative stress (DJ-1/GATase1), and energy status (AMPK/SnRK) pathways. They also showed statistical enrichment for genes in metabolic pathways that function in glycolysis, sugar degradation, inositol metabolism, and base excision repair. Collectively, the maize G4 motifs may represent conditional regulatory elements that can aid in energy status gene responses. Such a network of elements could provide a mechanistic basis for linking energy status signals to gene regulation in maize, a model genetic system and major world crop species for feed, food, and fuel.This article is from Journal of Genetics and Genomics 41 (2014): 627, doi: 10.1016/j.jgg.2014.10.004.</p

Structure and mechanism of B-family DNA polymerase ζ specialized for translesion DNA synthesis

DNA polymerase ζ (Polζ) belongs to the same B-family as high-fidelity replicative polymerases, yet is specialized for the extension reaction in translesion DNA synthesis (TLS). Despite its importance in TLS, the structure of Polζ is unknown. We present cryo-EM structures of the Saccharomyces cerevisiae Polζ holoenzyme in the act of DNA synthesis (3.1 Å) and without DNA (4.1 Å). Polζ displays a pentameric ring-like architecture, with catalytic Rev3, accessory Pol31' Pol32 and two Rev7 subunits forming an uninterrupted daisy chain of protein-protein interactions. We also uncover the features that impose high fidelity during the nucleotide-incorporation step and those that accommodate mismatches and lesions during the extension reaction. Collectively, we decrypt the molecular underpinnings of Polζ's role in TLS and provide a framework for new cancer therapeutics.This work was primarily funded by grant R01-GM124047 from the National Institutes of Health (NIH). I.U.-B. was supported by a grant PID2019-104423GB-I00/AEI/10.13039/501100011033 from the Spanish State Research Agency and by the Basque Excellence Research Centre program.Peer reviewe