A portable RNA sequence whose recognition by a synthetic antibody facilitates structural determination

Koldobskaya, Yelena; Duguid, E.M.; Shechner, David M; Suslov, N.B.; Ye, Jing‐Dong; Sidhu, Sachdev S.; Bartel, David P.; Koide, Shohei; Kossiakoff, Anthony A.; Piccirilli, Joseph A.

doi:10.1038/nsmb.1945

Cited by 81 publications

(143 citation statements)

References 53 publications

(71 reference statements)

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“…To do so, we biotinylated IDE in Escherichia coli, immobilized IDE on streptavidin-coated plates, used it to probe a phage-display library, and identified a synthetic antibody in the form of an antigen-binding fragment (Fab). This crystallization chaperone system has effectively facilitated crystallization of membrane proteins and RNA that are otherwise difficult to crystallize (23)(24)(25). The final synthetic antibody, termed Fab (IDE) , bound human IDE with a K D value of 4 nM, as determined by surface plasmon resonance (Fig.…”

Section: Resultsmentioning

confidence: 99%

Conformational states and recognition of amyloidogenic peptides of human insulin-degrading enzyme

McCord¹,

Liang²,

Dowdell

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Insulin-degrading enzyme (IDE) selectively degrades the monomer of amyloidogenic peptides and contributes to clearance of amyloid β (Aβ). Thus, IDE retards the progression of Alzheimer's disease. IDE possesses an enclosed catalytic chamber that engulfs and degrades its peptide substrates; however, the molecular mechanism of IDE function, including substrate access to the chamber and recognition, remains elusive. Here, we captured a unique IDE conformation by using a synthetic antibody fragment as a crystallization chaperone. An unexpected displacement of a door subdomain creates an ∼18-Å opening to the chamber. This swinging-door mechanism permits the entry of short peptides into the catalytic chamber and disrupts the catalytic site within IDE door subdomain. Given the propensity of amyloidogenic peptides to convert into β-strands for their polymerization into amyloid fibrils, they also use such β-strands to stabilize the disrupted catalytic site resided at IDE door subdomain for their degradation by IDE. Thus, action of the swinging door allows IDE to recognize amyloidogenicity by substrate-induced stabilization of the IDE catalytic cleft. Small angle X-ray scattering (SAXS) analysis revealed that IDE exists as a mixture of closed and open states. These open states, which are distinct from the swinging door state, permit entry of larger substrates (e.g., Aβ, insulin) to the chamber and are preferred in solution. Mutational studies confirmed the critical roles of the door subdomain and hinge loop joining the N-and C-terminal halves of IDE for catalysis. Together, our data provide insights into the conformational changes of IDE that govern the selective destruction of amyloidogenic peptides.M16 metalloprotease | X-ray crystallography | substrate recognition P roteins in living organisms face acute and chronic challenges to their integrity, which necessitate proteostatic processes to protect their functions (1). Protein-protease networks play a key role in proteostasis by ensuring proper protein function through protein turnovers (2). Amyloidogenic peptides, such as amyloid β (Aβ) and amylin, present a major challenge to proteostasis, because they can form toxic aggregates that impair diverse physiological functions and contribute to human diseases (3, 4). Insulin-degrading enzyme (IDE), a Zn 2+ -metalloprotease, prefers to degrade amyloidogenic peptides to prevent the formation of amyloid fibrils (3). Exemplary substrates of IDE are insulin and Aβ, which are critical for the development of type 2 diabetes mellitus (DM2) and Alzheimer's disease (AD), respectively. Genetic analyses strongly support functional roles of IDE in the clearance of insulin and Aβ (2, 3). In humans, several single nucleotide polymorphisms at the IDE locus on human chromosome 10q are associated with DM2 and late-onset AD (5, 6).Structural analyses have provided significant insights to substrate recognition and catalysis by IDE. IDE has two ∼50-kDa αβαβα N-terminal (IDE-N) and C-terminal (IDE-C) halves, which are linked by a short hinge loop...

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Section: Resultsmentioning

confidence: 99%

Conformational states and recognition of amyloidogenic peptides of human insulin-degrading enzyme

McCord¹,

Liang²,

Dowdell

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…The essential features of this method were sib selection strategy (McCormick 1987) combined with PCR mutagenesis using randomized codons of the nucleotide sequence NNS (N = A, G, C, or T; S = G or C). The previous affinity maturation study of anti-RNA antibodies screened from a synthetic library disclosed that mutations were observed in the CDR-L3 regions of higher-affinity antibodies at a relatively high frequency, rather than in other CDR regions (Koldobskaya et al 2011). Accordingly, CDR-L3 of FabBC200-A was chosen as an initial target CDR region for affinity maturation.…”

Section: Affinity Maturation Of the Anti-rna Antibodymentioning

confidence: 99%

“…1). In principle, we adapted the procedures used for the selection of RNA-binding proteins and synthetic antibodies from the groups of Belasco (Laird-Offringa and Belasco 1996) and Piccirilli (Ye et al 2008;Koldobskaya et al 2011), respectively. We used a streptavidin-coated immunotube and biotin-tagged BC200 RNA for RNA immobilization, which is known to facilitate more effective RNA binding and clearer separation of buffers than streptavidincoated beads.…”

Section: Fabs Selection Against Bc200 Rnamentioning

confidence: 99%

“…Experiments were performed as described previously (Koldobskaya et al 2011). Briefly, BC200 RNAs were 3 ′ end-labeled with [ 32 P]pCp using T4 RNA ligase (New England Biolabs).…”

Section: Rna Footprintingmentioning

confidence: 99%

“…Furthermore,4 nucleic acids such as RNA or DNA are not normally immunogenic, owing to recognition by immune cells as self-antigens (Pokkuluri et al 1994), although Stollar (1980) reported that antibodies to single-stranded DNA can be induced by linking them to proteins or polypeptides, followed by injecting to animals. On the other hand, anti-RNA antibodies can be obtained through panning and affinity maturation from an antibody library because previously Piccirilli's group reported the selection of specific antigen binding fragments (Fabs) against a domain derived from the Tetrahymena group I intron using a synthetic phage-display library (Ye et al 2008;Koldobskaya et al 2011).…”

Section: Introductionmentioning

confidence: 99%

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RNA recognition by a human antibody against brain cytoplasmic 200 RNA

Jung

Lee

Hong

et al. 2014

RNA

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Diverse functional RNAs participate in a wide range of cellular processes. The RNA structure is critical for function, either on its own or as a complex form with proteins and other ligands. Therefore, analysis of the RNA conformation in cells is essential for understanding their functional mechanisms. However, no appropriate methods have been established as yet. Here, we developed an efficient strategy for panning and affinity maturation of anti-RNA human monoclonal antibodies from a naïve antigen binding fragment (Fab) combinatorial phage library. Brain cytoplasmic 200 (BC200) RNA, which is also highly expressed in some tumors, was used as an RNA antigen. We identified MabBC200-A3 as the optimal binding antibody. Mutagenesis and SELEX experiments showed that the antibody recognized a domain of BC200 in a structure-and sequencedependent manner. Various breast cancer cell lines were further examined for BC200 RNA expression using conventional hybridization and immunoanalysis with MabBC200-A3 to see whether the antibody specifically recognizes BC200 RNA among the total purified RNAs. The amounts of antibody-recognizable BC200 RNA were consistent with hybridization signals among the cell lines. Furthermore, the antibody was able to discriminate BC200 RNA from other RNAs, supporting the utility of this antibody as a specific RNA structure-recognizing probe. Intriguingly, however, when permeabilized cells were subjected to immunoanalysis instead of purified total RNA, the amount of antibody-recognizable RNA was not correlated with the cellular level of BC200 RNA, indicating that BC200 RNA exists as two distinct forms (antibody-recognizable and nonrecognizable) in breast cancer cells and that their distribution depends on the cell type. Our results clearly demonstrate that anti-RNA antibodies provide an effective novel tool for detecting and analyzing RNA conformation.

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Use of a Novel 5′‐Regioselective Phosphitylating Reagent for One‐Pot Synthesis of Nucleoside 5′‐Triphosphates from Unprotected Nucleosides

Caton-Williams

Hoxhaj

Fiaz

et al. 2013

CP Nucleic Acid Chemistry

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The 5′-triphosphates are the building blocks for the enzymatic synthesis of DNAs and RNAs. This unit presents a protocol for the convenient synthesis of 2′-deoxyribo- and ribo-nucleoside 5′-triphosphates (dNTPs and NTPs) containing all the natural bases and the modified bases. This one-pot synthesis can also be applied to prepare the triphosphate analogs that contain sulfur or selenium atoms replacing the non-bridging oxygen atoms of the alpha phosphates of the triphosphates. These S- or Se-modified dNTPs and NTPs can be used to prepare diastereomerically-pure phosphorothioate nucleic acids (PS-NAs) or phosphoroselenoate nucleic acids (PSe-NAs, i.e., one type of selenium-derivatized nucleic acids: SeNA). Even without extensive purification, the synthesized dNTPs or NTPs are of high quality and can be directly used in DNA polymerization or RNA transcription. Synthesis and purification of the 5′-triphosphates, analysis and confirmation of natural and sulfur-or selenium-modified nucleic acids are described in this protocol unit.

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A portable RNA sequence whose recognition by a synthetic antibody facilitates structural determination

Cited by 81 publications

References 53 publications

Conformational states and recognition of amyloidogenic peptides of human insulin-degrading enzyme

Conformational states and recognition of amyloidogenic peptides of human insulin-degrading enzyme

RNA recognition by a human antibody against brain cytoplasmic 200 RNA

Use of a Novel 5′‐Regioselective Phosphitylating Reagent for One‐Pot Synthesis of Nucleoside 5′‐Triphosphates from Unprotected Nucleosides

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