Repertoire Of Structures Research Articles

2′-Azido RNA, a Versatile Tool for Chemical Biology: Synthesis, X-ray Structure, siRNA Applications, Click Labeling

Chemical modification can significantly enrich the structural and functional repertoire of ribonucleic acids and endow them with new outstanding properties. Here, we report the syntheses of novel 2′-azido cytidine and 2′-azido guanosine building blocks and demonstrate their efficient site-specific incorporation into RNA by mastering the synthetic challenge of using phosphoramidite chemistry in the presence of azido groups. Our study includes the detailed characterization of 2′-azido nucleoside containing RNA using UV-melting profile analysis and CD and NMR spectroscopy. Importantly, the X-ray crystallographic analysis of 2′-azido uridine and 2′-azido adenosine modified RNAs reveals crucial structural details of this modification within an A-form double helical environment. The 2′-azido group supports the C3′-endo ribose conformation and shows distinct water-bridged hydrogen bonding patterns in the minor groove. Additionally, siRNA induced silencing of the brain acid soluble protein (BASP1) encoding gene in chicken fibroblasts demonstrated that 2′-azido modifications are well tolerated in the guide strand, even directly at the cleavage site. Furthermore, the 2′-azido modifications are compatible with 2′-fluoro and/or 2′-O-methyl modifications to achieve siRNAs of rich modification patterns and tunable properties, such as increased nuclease resistance or additional chemical reactivity. The latter was demonstrated by the utilization of the 2′-azido groups for bioorthogonal Click reactions that allows efficient fluorescent labeling of the RNA. In summary, the present comprehensive investigation on site-specifically modified 2′-azido RNA including all four nucleosides provides a basic rationale behind the physico- and biochemical properties of this flexible and thus far neglected type of RNA modification.

Illuminating the functional and structural repertoire of human TBC/RABGAPs

The Tre2-Bub2-Cdc16 (TBC) domain-containing RAB-specific GTPase-activating proteins (TBC/RABGAPs) are characterized by the presence of highly conserved TBC domains and act as negative regulators of RABs. The importance of TBC/RABGAPs in the regulation of specific intracellular trafficking routes is now emerging, as is their role in different diseases. Importantly, TBC/RABGAPs act as key regulatory nodes, integrating signalling between RABs and other small GTPases and ensuring the appropriate retrieval, transport and delivery of different intracellular vesicles.

Disease mutations in disordered regions--exception to the rule?

Intrinsically disordered proteins (IDPs) have been implicated in a number of human diseases, including cancer, diabetes, neurodegenerative and cardiovascular disorders. Although for some of these conditions molecular mechanisms are now better understood, the big picture connecting distinct structural properties and functional repertoire of IDPs to pathogenesis and disease progression is still incomplete. Recent studies suggest that signaling and regulatory roles carried out by IDPs require them to be tightly regulated, and that altered IDP abundance may lead to disease. Here, we propose another link between IDPs and disease that takes into account disease-associated missense mutations located in the intrinsically disordered regions. We argue that such mutations are more prevalent and have larger functional impact than previously thought. In addition, we demonstrate that deleterious amino acid substitutions that cause disorder-to-order transitions are particularly enriched among disease mutations compared to neutral polymorphisms. Finally, we discuss potential differences in functional outcomes between disease mutations in ordered and disordered regions, and challenge the conventional structure-centric view of missense mutations.

Aqueous and Solvent‐Free Uncatalyzed Three‐Component Vinylogous Mukaiyama–Mannich Reactions of Pyrrole‐Based Silyl Dienolates

AbstractA catalyst‐free, environmentally benign three‐component vinylogous Mukaiyama–Mannich reaction of pyrrole‐based silyl dienolates is presented, which works effectively in both aqueous and solvent‐free environments. Both lipophilic and hydrophilic aldehyde candidates are suitable substrates, allowing access to a rich repertoire of unsaturated vicinal aminolactam structures with virtually complete γ‐site selectivity and moderate to good anti‐diastereoselectivity. The utility of this technology is highlighted by protecting group‐free synthesis of densely hydroxylated, sugar‐related lactam frameworks. The role of water as an indispensable H‐bonding reaction propeller is demonstrated.

A de novo peptide hexamer with a mutable channel

The design of new proteins that expand the repertoire of natural protein structures represents a formidable challenge. Success in this area would increase understanding of protein structure, and present new scaffolds that could be exploited in biotechnology and synthetic biology. Here we describe the design, characterisation and X-ray crystal structure of a new coiled-coil protein. The de novo sequence forms a stand-alone, parallel, 6-helix bundle with a channel running through it. Although lined exclusively by hydrophobic leucine and isoleucine side chains, the 6 Å channel is permeable to water. One layer of leucine residues within the channel is mutable accepting polar aspartic acid (Asp) and histidine (His) side chains, and leading to subdivision and organization of solvent within the lumen. Moreover, these mutants can be combined to form a stable and unique (Asp-His)3 heterohexamer. These new structures provide a basis for engineering de novo proteins with new functions.

Purification of Hsp104, a Protein Disaggregase

Hsp104 is a hexameric AAA+ protein(1) from yeast, which couples ATP hydrolysis to protein disaggregation (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure2). This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils. Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson's Disease as well as amyloid forms of PrP. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson's Disease and Huntington's disease. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors. To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102 kDa protein with a pI of -5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His(6)-tag removal compared to a previous purification method from E. coli. Moreover, our protocol is more facile and convenient than two more recent protocols.

Institutional Complexity and Organizational Responses

Organizations face institutional complexity whenever they confront incompatible prescriptions from multiple institutional logics. Our interest is in how plural institutional logics, refracted through field-level structures and processes, are experienced within organizations and how organizations respond to such complexity. We draw on a variety of cognate literatures to discuss the field-level structural characteristics and organizational attributes that shape institutional complexity. We then explore the repertoire of strategies and structures that organizations deploy to cope with multiple, competing demands. The analytical framework developed herein is presented to guide future scholarship in the systematic analysis of institutional complexity. We conclude by suggesting avenues for future research.

Institutional Complexity and Organizational Responses

Organizations face institutional complexity whenever they confront incompatible prescriptions from multiple institutional logics. Our interest is in how plural institutional logics, refracted through field-level structures and processes, are experienced within organizations and how organizations respond to such complexity. We draw on a variety of cognate literatures to discuss the field-level structural characteristics and organizational attributes that shape institutional complexity. We then explore the repertoire of strategies and structures that organizations deploy to cope with multiple, competing demands. The analytical framework developed herein is presented to guide future scholarship in the systematic analysis of institutional complexity. We conclude by suggesting avenues for future research.

Introducing the Korean Oligonucleotide Therapeutics Society

Nucleic Acid TherapeuticsVol. 21, No. 3 Articles from the Korean Oligonucleotide Therapeutics Society (K-OTS)Guest EditorialIntroducing the Korean Oligonucleotide Therapeutics SocietySeong-Wook LeeSeong-Wook LeeSearch for more papers by this authorPublished Online:29 Jun 2011https://doi.org/10.1089/nat.2011.1500AboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View article"Introducing the Korean Oligonucleotide Therapeutics Society." , 21(3), p. 123FiguresReferencesRelatedDetails Volume 21Issue 3Jun 2011 InformationCopyright 2011, Mary Ann Liebert, Inc.To cite this article:Seong-Wook Lee.Introducing the Korean Oligonucleotide Therapeutics Society.Nucleic Acid Therapeutics.Jun 2011.123-123.http://doi.org/10.1089/nat.2011.1500Published in Volume: 21 Issue 3: June 29, 2011PDF download

Bioinspiration: Applying Mechanical Design to Experimental Biology

The production of bioinspired and biomimetic constructs has fostered much collaboration between biologists and engineers, although the extent of biological accuracy employed in the designs produced has not always been a priority. Even the exact definitions of "bioinspired" and "biomimetic" differ among biologists, engineers, and industrial designers, leading to confusion regarding the level of integration and replication of biological principles and physiology. By any name, biologically-inspired mechanical constructs have become an increasingly important research tool in experimental biology, offering the opportunity to focus research by creating model organisms that can be easily manipulated to fill a desired parameter space of structural and functional repertoires. Innovative researchers with both biological and engineering backgrounds have found ways to use bioinspired models to explore the biomechanics of organisms from all kingdoms to answer a variety of different questions. Bringing together these biologists and engineers will hopefully result in an open discourse of techniques and fruitful collaborations for experimental and industrial endeavors.

Combinations of Protein-Chemical Complex Structures Reveal New Targets for Established Drugs

Biological networks are powerful tools for predicting undocumented relationships between molecules. The underlying principle is that existing interactions between molecules can be used to predict new interactions. Here we use this principle to suggest new protein-chemical interactions via the network derived from three-dimensional structures. For pairs of proteins sharing a common ligand, we use protein and chemical superimpositions combined with fast structural compatibility screens to predict whether additional compounds bound by one protein would bind the other. The method reproduces 84% of complexes in a benchmark, and we make many predictions that would not be possible using conventional modeling techniques. Within 19,578 novel predicted interactions are 7,793 involving 718 drugs, including filaminast, coumarin, alitretonin and erlotinib. The growth rate of confident predictions is twice that of experimental complexes, meaning that a complete structural drug-protein repertoire will be available at least ten years earlier than by X-ray and NMR techniques alone.

Structural repertoire of immunoglobulin λ light chains

The immunoglobulin λ isotype is present in nearly all vertebrates and plays an important role in the human immune system. Despite its importance, few systematic studies have been performed to analyze the structural conformation of its variable regions, contrary to what is the case for κ and heavy chains. We show here that an analysis of the structures of λ chains allows the definition of a discrete set of recurring conformations (canonical structures) of their hypervariable loops and, most importantly, the identification of sequence constraints that can be used to predict their structure. We also show that the structural repertoire of λ chains is different and more varied than that of the κ chains, consistently with the current view of the involvement of the two major light-chain families in complementary strategies of the immune system to ensure a fine tuning between diversity and stability in antigen recognition.

Genome analysis and comparative genomics of a Giardia intestinalis assemblage E isolate

BackgroundGiardia intestinalis is a protozoan parasite that causes diarrhea in a wide range of mammalian species. To further understand the genetic diversity between the Giardia intestinalis species, we have performed genome sequencing and analysis of a wild-type Giardia intestinalis sample from the assemblage E group, isolated from a pig.ResultsWe identified 5012 protein coding genes, the majority of which are conserved compared to the previously sequenced genomes of the WB and GS strains in terms of microsynteny and sequence identity. Despite this, there is an unexpectedly large number of chromosomal rearrangements and several smaller structural changes that are present in all chromosomes. Novel members of the VSP, NEK Kinase and HCMP gene families were identified, which may reveal possible mechanisms for host specificity and new avenues for antigenic variation. We used comparative genomics of the three diverse Giardia intestinalis isolates P15, GS and WB to define a core proteome for this species complex and to identify lineage-specific genes. Extensive analyses of polymorphisms in the core proteome of Giardia revealed differential rates of divergence among cellular processes.ConclusionsOur results indicate that despite a well conserved core of genes there is significant genome variation between Giardia isolates, both in terms of gene content, gene polymorphisms, structural chromosomal variations and surface molecule repertoires. This study improves the annotation of the Giardia genomes and enables the identification of functionally important variation.

Role of the Conformational Versatility of the Neurotrophin N-Terminal Regions in Their Recognition by Trk Receptors

Neurotrophins (NTs) represent a family of proteins that play an important role in the survival, development, and function of neurons. Extensive efforts are currently being made to develop small molecules endowed with agonist or antagonist NT activity. The structurally versatile N-termini of these proteins are considered regions of interest for the design of new molecules. By combining experimental and computational approaches, we analyzed the intrinsic conformational preferences of the N-termini of two of the most important NTs: NGF (NGF-Nter) and NT4 (NT4-Nter). Circular dichroism spectra clearly indicate that both peptides show a preference for random coil states. Because this finding does not preclude the possibility that structured forms may occur in solution as minor conformational states, we performed molecular-dynamics simulations to gain insights into the structural features of populated species. In line with the circular dichroism analysis, the simulations show a preference for unstructured states for both peptides. However, the simulations also show that for NT4-Nter, and to a lesser extent for NGF-Nter, helical conformations, which are required for binding to the Trk receptor, are present in the repertoire of structures that are intrinsically accessible to these peptides. Accordingly, molecular recognition of NTs by the Trk receptor is accomplished by the general mechanism known as population shift. These findings provide a structural rationale for the observed activity of synthetic peptides based on these NT regions. They also suggest strategies for the development of biologically active peptide-based compounds.

Complementary Roles for Exonuclease 1 and Flap Endonuclease 1 in Maintenance of Triplet Repeats

Trinucleotide repeats can form stable secondary structures that promote genomic instability. To determine how such structures are resolved, we have defined biochemical activities of the related RAD2 family nucleases, FEN1 (Flap endonuclease 1) and EXO1 (exonuclease 1), on substrates that recapitulate intermediates in DNA replication. Here, we show that, consistent with its function in lagging strand replication, human (h) FEN1 could cleave 5'-flaps bearing structures formed by CTG or CGG repeats, although less efficiently than unstructured flaps. hEXO1 did not exhibit endonuclease activity on 5'-flaps bearing structures formed by CTG or CGG repeats, although it could excise these substrates. Neither hFEN1 nor hEXO1 was affected by the stem-loops formed by CTG repeats interrupting duplex regions adjacent to 5'-flaps, but both enzymes were inhibited by G4 structures formed by CGG repeats in analogous positions. Hydroxyl radical footprinting showed that hFEN1 binding caused hypersensitivity near the flap/duplex junction, whereas hEXO1 binding caused hypersensitivity very close to the 5'-end, correlating with the predominance of hFEN1 endonucleolytic activity versus hEXO1 exonucleolytic activity on 5'-flap substrates. These results show that FEN1 and EXO1 can eliminate structures formed by trinucleotide repeats in the course of replication, relying on endonucleolytic and exonucleolytic activities, respectively. These results also suggest that unresolved G4 DNA may prevent key steps in normal post-replicative DNA processing.

680 Epigenetic silencing of miR-203 is a disease initiation event of multiple myeloma

Protocadherins are predominantly expressed in the nervous system, and constitute the largest subgroup within the cadherin superfamily. The recent structural elucidation of the amino-terminal cadherin domain in an archetypal protocadherin revealed unique and remarkable features: the lack of an interface for homophilic adhesiveness found in classical cadherins, and the presence of loop structures specific to the protocadherin family. The unique features of protocadherins extend to their genomic organization. Recent findings have revealed unexpected allelic and combinatorial gene regulation for clustered protocadherins, a major subgroup in the protocadherin family. The unique structural repertoire and unusual gene regulation of the protocadherin family may provide the molecular basis for the extraordinary diversity of the nervous system.

The Genome Sequence of Trypanosoma brucei gambiense, Causative Agent of Chronic Human African Trypanosomiasis

Background Trypanosoma brucei gambiense is the causative agent of chronic Human African Trypanosomiasis or sleeping sickness, a disease endemic across often poor and rural areas of Western and Central Africa. We have previously published the genome sequence of a T. b. brucei isolate, and have now employed a comparative genomics approach to understand the scale of genomic variation between T. b. gambiense and the reference genome. We sought to identify features that were uniquely associated with T. b. gambiense and its ability to infect humans.Methods and FindingsAn improved high-quality draft genome sequence for the group 1 T. b. gambiense DAL 972 isolate was produced using a whole-genome shotgun strategy. Comparison with T. b. brucei showed that sequence identity averages 99.2% in coding regions, and gene order is largely collinear. However, variation associated with segmental duplications and tandem gene arrays suggests some reduction of functional repertoire in T. b. gambiense DAL 972. A comparison of the variant surface glycoproteins (VSG) in T. b. brucei with all T. b. gambiense sequence reads showed that the essential structural repertoire of VSG domains is conserved across T. brucei.ConclusionsThis study provides the first estimate of intraspecific genomic variation within T. brucei, and so has important consequences for future population genomics studies. We have shown that the T. b. gambiense genome corresponds closely with the reference, which should therefore be an effective scaffold for any T. brucei genome sequence data. As VSG repertoire is also well conserved, it may be feasible to describe the total diversity of variant antigens. While we describe several as yet uncharacterized gene families with predicted cell surface roles that were expanded in number in T. b. brucei, no T. b. gambiense-specific gene was identified outside of the subtelomeres that could explain the ability to infect humans.

Caracterização genética de espécies de Passiflora por marcadores moleculares análogos a genes de resistência

O cultivo comercial do maracujá é afetado por diversos problemas fitossanitários, os quais contribuem para quebras de produção e significativa redução da vida útil dos plantios. Em algumas situações, a incidência de doenças pode inviabilizar o cultivo do maracujá. Fontes de resistência a distintas doenças têm sido identificadas em acessos de espécies de Passiflora. Neste trabalho, buscou-se avaliar a diversidade genética de acessos de oito espécies silvestres (P. setacea, P. nitida, P. serratodigitata, P. caerulea, P. gibertii, P. odontophyla, P. edulis e P. coccinea) e de um híbrido interespecífico (P. setacea x P. coccinea), utilizando marcadores moleculares análogos a genes de resistência (RGAs). Verificou-se uma grande diversidade no perfil eletroforético de RGAs nos acessos de Passiflora, permitindo a anotação de 96 amplicons polimórficos entre, pelo menos, um par de acessos. Os níveis de dissimilaridade genética (calculados exclusivamente com os marcadores RGAs) variaram entre 0,40 e 0,89 nos acessos das espécies de Passiflora avaliadas. A análise de sequência de um subgrupo destes amplicons obtidos com primers RGAs indicou que estas bandas correspondem a regiões genômicas que contêm segmentos (motivos) com identidade aos encontrados em genes de resistência previamente caracterizados em outras espécies vegetais. Desta forma, os dados indicam a existência de um repertório variado de marcadores do tipo RGA em Passiflora que podem ser potencialmente úteis em sistemas de caracterização molecular de germoplasma e em programas de melhoramento genético visando à resistência a doenças nesta cultura.

Structural and kinetic determinants of protease substrates

Two fundamental questions with regard to proteolytic networks and pathways concern the structural repertoire and kinetic threshold that distinguish legitimate signaling substrates. We used N-terminal proteomics to address these issues by identifying cleavage sites within the Escherichia coli proteome that are driven by the apoptotic signaling protease caspase-3 and the bacterial protease glutamyl endopeptidase (GluC). Defying the dogma that proteases cleave primarily in natively unstructured loops, we found that both caspase-3 and GluC cleave in alpha-helices nearly as frequently as in extended loops. Notably, biochemical and kinetic characterization revealed that E. coli caspase-3 substrates are greatly inferior to natural substrates, suggesting protease and substrate coevolution. Engineering an E. coli substrate to match natural catalytic rates defined a kinetic threshold that depicts a signaling event. This unique combination of proteomics, biochemistry, kinetics and substrate engineering reveals new insights into the structure-function relationship of protease targets and their validation from large-scale approaches.

Constraint on the Number of Synaptic Inputs to a Visual Cortical Neuron Controls Receptive Field Formation

To date, Hebbian learning combined with some form of constraint on synaptic inputs has been demonstrated to describe well the development of neural networks. The previous models revealed mathematically the importance of synaptic constraints to reproduce orientation selectivity in the visual cortical neurons, but biological mechanisms underlying such constraints remain unclear. In this study, we addressed this issue by formulating a synaptic constraint based on activity-dependent mechanisms of synaptic changes. Particularly, considering metabotropic glutamate receptor-mediated long-term depression, we derived synaptic constraint that suppresses the number of inputs from individual presynaptic neurons. We performed computer simulations of the activity-dependent self-organization of geniculocortical inputs with the synaptic constraint and examined the formation of receptive fields (RFs) of model visual cortical neurons. When we changed the magnitude of the synaptic constraint, we found the emergence of distinct RF structures such as concentric RFs, simple-cell-like RFs, and double-oriented RFs and also a gradual transition between spatiotemporal separable and inseparable RFs. Thus, the model based on the synaptic constraint derived from biological consideration can account systematically for the repertoire of RF structures observed in the primary visual cortices of different species for the first time.