Protein Structure and Folding
Structural delineation and phase-dependent activation of the costimulatory CD27:CD70 complex
CD27 is a tumor necrosis factor (TNF) receptor, which stimulates lymphocytes and promotes their differentiation upon activation by TNF ligand CD70. Activation of the CD27 receptor provides a costimulatory signal to promote T cell, B cell, and NK cell activity to facilitate antitumor and anti-infection immunity. Aberrant increased and focused expression of CD70 on many tumor cells renders CD70 an attractive therapeutic target for direct tumor killing. However, despite their use as drug targets to treat cancers, the molecular basis and atomic details of CD27 and CD70 interaction remain elusive.Structural optimization of a TNFR1-selective antagonistic TNFα mutant to create new-modality TNF-regulating biologics
Excessive activation of the proinflammatory cytokine tumor necrosis factor-α (TNFα) is a major cause of autoimmune diseases, including rheumatoid arthritis. TNFα induces immune responses via TNF receptor 1 (TNFR1) and TNFR2. Signaling via TNFR1 induces proinflammatory responses, whereas TNFR2 signaling is suggested to suppress the pathophysiology of inflammatory diseases. Therefore, selective inhibition of TNFR1 signaling and preservation of TNFR2 signaling activities may be beneficial for managing autoimmune diseases.Specificity from nonspecific interaction: regulation of tumor necrosis factor-α activity by DNA
As anionic biopolymers, oligonucleotides can have biological functions independent from their roles as the medium for the storage and flow of genetic information. In this paper, we investigated the interaction between DNA and the pro-inflammatory cytokine tumor necrosis factor-α (TNFα). Although various forms of DNA bind to TNFα with low μm dissociation constants, the interaction stabilizes the trimeric form of TNFα and enhances its cytotoxic effect. Based on this mechanism, a photoswitchable TNFα (TNFα–2-nitroveratryloxycarbonyl) has been designed whose sensitivity to DNA-mediated up-regulation of TNFα activity can be tuned by light irradiation.Overexpression of the C-domain of angiotensin-converting enzyme reduces melanoma growth by stimulating M1 macrophage polarization
Angiotensin-converting enzyme (ACE) can hydrolyze many peptides and plays a central role in controlling blood pressure. Moreover, ACE overexpression in monocytes and macrophages increases resistance of mice to tumor growth. ACE is composed of two independent catalytic domains. Here, to investigate the specific role of each domain in tumor resistance, we overexpressed either WT ACE (Tg-ACE mice) or ACE lacking N- or C-domain catalytic activity (Tg-NKO and Tg-CKO mice) in the myeloid cells of mice.Crystal structures of the human 4-1BB receptor bound to its ligand 4-1BBL reveal covalent receptor dimerization as a potential signaling amplifier
Human (h)4-1BB (TNFRSF9 or CD137) is an inducible tumor necrosis factor receptor (TNFR) superfamily member that interacts with its cognate ligand h4-1BBL to promote T lymphocyte activation and proliferation. h4-1BB is currently being targeted with agonists in cancer immunotherapy. Here, we determined the crystal structures of unbound h4-1BBL and both WT h4-1BB and a dimerization-deficient h4-1BB mutant (C121S) in complex with h4-1BBL at resolutions between 2.7 and 3.2 Å. We observed that the structural arrangement of 4-1BBL, both unbound and in the complex, represents the canonical bell shape as seen in other similar TNF proteins and differs from the previously reported three-bladed propeller structure of 4-1BBL.Crystal structure of the human 4-1BB/4-1BBL complex
4-1BBL is a member of the tumor necrosis factor (TNF) superfamily and is the ligand for the TNFR superfamily receptor, 4-1BB. 4-1BB plays an immunomodulatory role in T cells and NK cells, and agonists of this receptor have garnered strong attention as potential immunotherapy agents. Broadly speaking, the structural features of TNF superfamily members, their receptors, and ligand-receptor complexes are similar. However, a published crystal structure of human 4-1BBL suggests that it may be unique in this regard, exhibiting a three-bladed propeller-like trimer assembly that is distinctly different from that observed in other family members.Crystal structure of murine 4-1BB and its interaction with 4-1BBL support a role for galectin-9 in 4-1BB signaling
4-1BB (CD137) is a TNF receptor superfamily (TNFRSF) member that is thought to undergo receptor trimerization upon binding to its trimeric TNF superfamily ligand (4-1BBL) to stimulate immune responses. 4-1BB also can bind to the tandem repeat–type lectin galectin-9 (Gal-9), and signaling through mouse (m)4-1BB is reduced in galectin-9 (Gal-9)–deficient mice, suggesting a pivotal role of Gal-9 in m4-1BB activation. Here, using sulfur-SAD phasing, we determined the crystal structure of m4-1BB to 2.2-Å resolution.Structure of the human aminopeptidase XPNPEP3 and comparison of its in vitro activity with Icp55 orthologs: Insights into diverse cellular processes
The human aminopeptidase XPNPEP3 is associated with cystic kidney disease and TNF-TNFR2 cellular signaling. Its yeast and plant homolog Icp55 processes several imported mitochondrial matrix proteins leading to their stabilization. However, the molecular basis for the diverse roles of these enzymes in the cell is unknown. Here, we report the crystal structure of human XPNPEP3 with bound apstatin product at 1.65 Å resolution, and we compare its in vitro substrate specificity with those of fungal Icp55 enzymes.A trimeric structural fusion of an antagonistic tumor necrosis factor-α mutant enhances molecular stability and enables facile modification
Tumor necrosis factor-α (TNF) exerts its biological effect through two types of receptors, p55 TNF receptor (TNFR1) and p75 TNF receptor (TNFR2). An inflammatory response is known to be induced mainly by TNFR1, whereas an anti-inflammatory reaction is thought to be mediated by TNFR2 in some autoimmune diseases. We have been investigating the use of an antagonistic TNF mutant (TNFR1-selective antagonistic TNF mutant (R1antTNF)) to reveal the pharmacological effect of TNFR1-selective inhibition as a new therapeutic modality.Fc Engineering Approaches to Enhance the Agonism and Effector Functions of an Anti-OX40 Antibody
Agonistic antibodies directed against immunostimulatory receptors belonging to the tumor necrosis factor receptor (TNFR) superfamily are emerging as promising cancer immunotherapies. Several Fc engineering approaches discovered recently can augment the anti-tumor activities of TNFR antibodies by enhancing their agonistic activities and/or effector functions. In this study, we compared these approaches for their effects on an anti-OX40 antibody. Both S267E/L328F and V12 mutations facilitated enhanced binding to FcγRIIB and thus increased FcγRIIB cross-linking mediated agonist activity.The Lectin-like Domain of TNF Increases ENaC Open Probability through a Novel Site at the Interface between the Second Transmembrane and C-terminal Domains of the α-Subunit
Regulation of the epithelial sodium channel (ENaC), which regulates fluid homeostasis and blood pressure, is complex and remains incompletely understood. The TIP peptide, a mimic of the lectin-like domain of TNF, activates ENaC by binding to glycosylated residues in the extracellular loop of ENaC-α, as well as to a hitherto uncharacterized internal site. Molecular docking studies suggested three residues, Val567, Glu568, and Glu571, located at the interface between the second transmembrane and C-terminal domains of ENaC-α, as a critical site for binding of the TIP peptide.Rapid Fine Conformational Epitope Mapping Using Comprehensive Mutagenesis and Deep Sequencing
Background: A new method using comprehensive mutagenesis libraries, yeast display, and deep sequencing is proposed to determine fine conformational epitopes for three antibody-antigen interactions.Results: For three separate antigens, the experimentally determined conformational epitope is consistent with orthogonal experimental datasets.Conclusion: We conclude that this new methodology is reliable and sound.Significance: With this new method, four antibody-antigen interactions can be mapped per day.
