Subtle changes at the variable domain interface of the T-cell receptor can strongly increase affinity [Immunology]

December 14th, 2017 by Preeti Sharma, David M Kranz

Most affinity-maturation campaigns for antibodies and T cell receptors (TCRs) operate on the residues at the binding site, located within the loops known as complementarity determining regions (CDRs). Accordingly, mutations in contact residues, or so-called "second shell" residues, that increase affinity are typically identified by group at the α:β interface, at a significant distance from the TCR/pepMHC binding site, remarkably affected ligand binding. The variant retained a high degree of specificity for MART- 1/HLA-A2, indicating that our approach provides a general strategy for engineering improvements in either soluble or cell-based TCRs for therapeutic purposes.directed evolution involving combinatorial libraries. To determine the impact of residues located at a distance from the binding site, here we used single codon libraries of both CDR and non- CDR residues to generate a deep mutational scan of a human TCR against the cancer antigen MART-1/HLA-A2. Non-CDR residues included those at the interface of the TCR variable domains (α and β) and surface-exposed framework residues. Mutational analyses showed that both α:β interface and CDR residues were important in maintaining binding to MART- 1/HLA-A2, likely due to either structural requirements for proper α:β association or direct contact with the ligand. More surprisingly, many α:β interface substitutions yielded improved binding to MART-1/HLA-A2. To further explore this finding, we constructed interface libraries and selected them for improved stability or affinity. Among the variants identified, one conservative substitution (β) was most prevalent. Further analysis of β showed that it enhanced thermostability and increased affinity by 60-fold. Thus, introducing a single hydroxyl group at the α:β interface, at a significant distance from the TCR/pepMHC binding site, remarkably affected ligand binding. The variant retained a high degree of specificity for MART- 1/HLA-A2, indicating that our approach provides a general strategy for engineering improvements in either soluble or cell-based TCRs for therapeutic purposes.

The inducible microRNA-203 in fish represses the inflammatory responses to Gram-negative bacteria by targeting IL-1 receptor-associated kinase 4 [Gene Regulation]

December 14th, 2017 by Tianjun Xu, Qing Chu, Junxia Cui, Xueyan Zhao

Innate immune responses are the first defense against pathogenic invaders. Activation and termination of these immune responses are regulated by several mechanisms. MicroRNAs (miRNAs), a group of small non-coding RNAs, have been implicated in the regulation of a spectrum of both physiological and pathological conditions, including immune responses. Although immune regulatory miRNAs networks in higher vertebrates have been well described, regulation of these responses in fish species is poorly understood in fish species. In the present study, we investigated the role of the miRNA miR-203 involved in inflammatory responses in miiuy croaker (Miichthys miiuy). We found that the Gram-negative bacterium, Vibrio anguillarum and LPS significantly upregulated host miR-203 expression. The increased miR-203 expression suppressed the production of inflammatory cytokines and thereby prevented mounting of a full immune response. Mechanistically, we identified and validated IL-1 receptor-associated kinase 4 (IRAK4) as a target of miR-203. We observed that miR-203 could post-transcriptionally controls IRAK4 expression and thereby inhibits the activation of nuclear factor kappa B (NF-κB) signaling. In summary, our findings reveal that miR-203 in fish is a critical suppressor of innate immune responses to bacterial infection by suppressing a feedback to IRAK4-NF-κB-mediated signaling in fish.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on The inducible microRNA-203 in fish represses the inflammatory responses to Gram-negative bacteria by targeting IL-1 receptor-associated kinase 4 [Gene Regulation]

RNA modifications: Ribosomes get decorated

December 12th, 2017 by Mirella Bucci

RNA modifications: Ribosomes get decorated

RNA modifications: Ribosomes get decorated, Published online: 12 December 2017; doi:10.1038/nchembio.2543

RNA modifications: Ribosomes get decorated

Notch signaling: A sweet strategy

December 12th, 2017 by Tetsuya Okajima

Notch signaling: A sweet strategy

Notch signaling: A sweet strategy, Published online: 12 December 2017; doi:10.1038/nchembio.2532

Glycosylation of Notch receptors regulates ligand-induced Notch signaling, which is essential for normal development in animals. Fucose analogs targeting Notch glycosylation serve as ligand-specific Notch inhibitors and facilitate the understanding of how O-glycan regulates Notch–ligand interactions.

Microbiology: Trapping Rac1

December 12th, 2017 by Karin Kuehnel

Microbiology: Trapping Rac1

Microbiology: Trapping Rac1, Published online: 12 December 2017; doi:10.1038/nchembio.2541

Microbiology: Trapping Rac1

Resistance mechanisms: Watering down a warhead

December 12th, 2017 by Caitlin Deane

Resistance mechanisms: Watering down a warhead

Resistance mechanisms: Watering down a warhead, Published online: 12 December 2017; doi:10.1038/nchembio.2542

Resistance mechanisms: Watering down a warhead

Protein engineering: Finding the best ligase

December 12th, 2017 by Christian F W Becker

Protein engineering: Finding the best ligase

Protein engineering: Finding the best ligase, Published online: 12 December 2017; doi:10.1038/nchembio.2533

Modification of folded proteins at will, within any sequence context, remains an elusive goal. A proteome-wide screening approach has now identified a set of protein ligases that enables conjugation of peptides to almost any protein N terminus, overcoming longstanding limitations in protein engineering.

Neurobiology: Defining your territory

December 12th, 2017 by Grant Miura

Neurobiology: Defining your territory

Neurobiology: Defining your territory, Published online: 12 December 2017; doi:10.1038/nchembio.2544

Neurobiology: Defining your territory

Structure-inspired design of β-arrestin-biased ligands for aminergic GPCRs

December 11th, 2017 by John D McCorvy

Structure-inspired design of β-arrestin-biased ligands for aminergic GPCRs

Structure-inspired design of β-arrestin-biased ligands for aminergic GPCRs, Published online: 11 December 2017; doi:10.1038/nchembio.2527

Development of D2 dopamine receptor ligands biased for β-arrestin recruitment based on a receptor homology model that identified conserved ligand contacts within the TM5 and EL2 regions as important for biased signaling.
  • Posted in Nat Chem Biol, Publications
  • Comments Off on Structure-inspired design of β-arrestin-biased ligands for aminergic GPCRs

Engineered synthetic scaffolds for organizing proteins within the bacterial cytoplasm

December 11th, 2017 by Matthew J Lee

Engineered synthetic scaffolds for organizing proteins within the bacterial cytoplasm

Engineered synthetic scaffolds for organizing proteins within the bacterial cytoplasm, Published online: 11 December 2017; doi:10.1038/nchembio.2535

Two complementary coiled-coil peptides and a bacterial microcompartment shell protein are combined to construct cytoscaffolds within Escherichia coli cells. Targeting enzymes to the cytoplasmic scaffold results in colocalization and improved metabolic flux.
  • Posted in Nat Chem Biol, Publications
  • Comments Off on Engineered synthetic scaffolds for organizing proteins within the bacterial cytoplasm