Preclinical anti-tumour activity of HexaBody-CD38, a next-generation CD38 antibody with superior complement-dependent cytotoxic activity.

Background: HexaBody®-CD38 (GEN3014) is a hexamerization-enhanced human IgG1 that binds CD38 with high affinity. The E430G mutation in its Fc domain facilitates the natural process of antibody hexamer formation upon binding to the cell surface, resulting in increased binding of C1q and potentiated complement-dependent cytotoxicity (CDC).

Methods: Co-crystallization studies were performed to identify the binding interface of HexaBody-CD38 and CD38.

Comparison of methods generating antibody-epitope conjugates for targeting cancer with virus-specific T cells.

Therapeutic antibody-epitope conjugates (AECs) are promising new modalities to deliver immunogenic epitopes and redirect virus-specific T-cell activity to cancer cells. Nevertheless, many aspects of these antibody conjugates require optimization to increase their efficacy. Here we evaluated different strategies to conjugate an EBV epitope (YVL/A2) preceded by a protease cleavage site to the antibodies cetuximab and trastuzumab.

Dual Epitope Targeting and Enhanced Hexamerization by DR5 Antibodies as a Novel Approach to Induce Potent Antitumor Activity Through DR5 Agonism.

Higher-order death receptor 5 (DR5) clustering can induce tumor cell death; however, therapeutic compounds targeting DR5 have achieved limited clinical efficacy. We describe HexaBody-DR5/DR5, an equimolar mixture of two DR5-specific IgG1 antibodies with an Fc-domain mutation that augments antibody hexamerization after cell surface target binding. The two antibodies do not compete for binding to DR5 as demonstrated using binding competition studies, and binding to distinct epitopes in the DR5 extracellular domain was confirmed by crystallography.

Direct binding of Cdt2 to PCNA is important for targeting the CRL4 E3 ligase activity to Cdt1.

The CRL4 ubiquitin ligase complex is an essential regulator of cell-cycle progression and genome stability, ubiquitinating substrates such as p21, Set8, and Cdt1, via a display of substrate degrons on proliferating cell nuclear antigens (PCNAs). Here, we examine the hierarchy of the ligase and substrate recruitment kinetics onto PCNA at sites of DNA replication. We demonstrate that the C-terminal end of Cdt2 bears a PCNA interaction protein motif (PIP box, Cdt2), which is necessary and sufficient for the binding of Cdt2 to PCNA.

Ca2+-dependent structural changes in the B-cell receptor CD23 increase its affinity for human immunoglobulin E.

Immunoglobulin E (IgE) antibodies play a fundamental role in allergic disease and are a target for therapeutic intervention. IgE functions principally through two receptors, FcεRI and CD23 (FcεRII). Minute amounts of allergen trigger mast cell or basophil degranulation by cross-linking IgE-bound FcεRI, leading to an inflammatory response.

Intrinsic flexibility of ubiquitin on proliferating cell nuclear antigen (PCNA) in translesion synthesis.

Ubiquitin conjugation provides a crucial signaling role in hundreds of cellular pathways; however, a structural understanding of ubiquitinated substrates is lacking. One important substrate is monoubiquitinated PCNA (PCNA-Ub), which signals for recruitment of damage-tolerant polymerases in the translesion synthesis (TLS) pathway of DNA damage avoidance. We use a novel and efficient enzymatic method to produce PCNA-Ub at high yield with a native isopeptide bond and study its Usp1/UAF1-dependent deconjugation.

Mapping of the CD23 binding site on immunoglobulin E (IgE) and allosteric control of the IgE-Fc epsilonRI interaction.

IgE, the antibody that mediates allergic responses, acts as part of a self-regulating protein network. Its unique effector functions are controlled through interactions of its Fc region with two cellular receptors, FcεRI on mast cells and basophils and CD23 on B cells. IgE cross-linked by allergen triggers mast cell activation via FcεRI, whereas IgE-CD23 interactions control IgE expression levels.

Human RAD18 interacts with ubiquitylated chromatin components and facilitates RAD9 recruitment to DNA double strand breaks.

RAD18 is an ubiquitin ligase involved in replicative damage bypass and DNA double-strand break (DSB) repair processes. We found that RPA is required for the dynamic pattern of RAD18 localization during the cell cycle, and for accumulation of RAD18 at sites of γ-irradiation-induced DNA damage. In addition, RAD18 colocalizes with chromatin-associated conjugated ubiquitin and ubiquitylated H2A throughout the cell cycle and following irradiation.

Symmetry and asymmetry of the RING-RING dimer of Rad18.

The human ubiquitin-conjugating enzyme Rad6 (E2), with ubiquitin ligase enzyme Rad18 (RING E3), monoubiquitinates proliferating cell nuclear antigen at stalled replication forks in DNA translesion synthesis. Here, we determine the structure of the homodimeric Rad18 RING domains by X-ray crystallography and classify it to RING-RING dimers that dimerize through helices adjacent to the RING domains and through the canonical RING domains. Using NMR spectroscopy and site-directed mutagenesis, we demonstrate that the Rad6b binding site, for the Rad18 RING domain, strongly resembles that of other E2/E3 RING/U-box complexes.

E3 ligase Rad18 promotes monoubiquitination rather than ubiquitin chain formation by E2 enzyme Rad6.

In ubiquitin conjugation, different combinations of E2 and E3 enzymes catalyse either monoubiquitination or ubiquitin chain formation. The E2/E3 complex Rad6/Rad18 exclusively monoubiquitinates the proliferating cell nuclear antigen (PCNA) to signal for "error prone" DNA damage tolerance, whereas a different set of conjugation enzymes is required for ubiquitin chain formation on PCNA. Here we show that human E2 enzyme Rad6b is intrinsically capable of catalyzing ubiquitin chain formation.

Regulation of translesion synthesis DNA polymerase eta by monoubiquitination.

DNA polymerase eta is a Y family polymerase involved in translesion synthesis (TLS). Its action is initiated by simultaneous interaction between the PIP box in pol eta and PCNA and between the UBZ in pol eta and monoubiquitin attached to PCNA. Whereas monoubiquitination of PCNA is required for its interaction with pol eta during TLS, we now show that monoubiquitination of pol eta inhibits this interaction, preventing its functions in undamaged cells.

Structural aspects of multi-domain RING/Ubox E3 ligases in DNA repair.

Ubiquitin conjugation plays critical roles in virtually all DNA repair pathways. This review provides an overview of the known multi-domain RING/Ubox E3 ligases and their domain structures. An analysis of known RING/Ubox X-ray and NMR structures leads to a discussion of the effects of dimerization.

Human Wrnip1 is localized in replication factories in a ubiquitin-binding zinc finger-dependent manner.

Wrnip1 (Werner helicase-interacting protein 1) has been implicated in the bypass of stalled replication forks in bakers' yeast. However, the function(s) of human Wrnip1 has remained elusive so far. Here we report that Wrnip1 is distributed inside heterogeneous structures detectable in nondamaged cells throughout the cell cycle.

Functional characterization of Rad18 domains for Rad6, ubiquitin, DNA binding and PCNA modification.

Rad18 is a ubiquitin E3 ligase that monoubiquitinates PCNA on stalled replications forks. This allows recruitment of damage-tolerant polymerases for damage bypass and DNA repair. In this activity, the Rad18 protein has to interact with Rad6, the E2 ubiquitin-conjugating enzyme, ubiquitin, PCNA and DNA.

Creating Mechanisms for Meaningful Collaboration Between Members of Urban Communities and University-Based HIV Prevention Researchers.

This article provides a description of a Community/University Collaborative Board, a formalized partnership between representatives from an inner-city community and university-based researchers. This Collaborative Board oversees a number of research projects focused on designing, delivering and testing family-based HIV prevention and mental health focused programs to elementary and junior high school age youth and their families. The Collaborative Board consists of urban parents, school staff members, representatives from community-based agencies and university-based researchers.

The structure of human CD23 and its interactions with IgE and CD21.

The low-affinity immunoglobulin E (IgE) receptor, CD23 (FcepsilonRII), binds both IgE and CD21 and, through these interactions, regulates the synthesis of IgE, the antibody isotype that mediates the allergic response. We have determined the three-dimensional structure of the C-type lectin domain of CD23 in solution by nuclear magnetic resonance spectroscopy. An analysis of concentration-dependent chemical shift perturbations have allowed us to identify the residues engaged in self-association to the trimeric state, whereas ligand-induced changes have defined the binding sites for IgE and CD21.

Transition metal-directed self-assembly of calix[4]arene based dithiocarbamate ligands.

The transition metal-directed self-assembly of dithiocarbamate ligand functionalized upper and lower rim calix[4]arenes affords novel dimeric bimetallic bis(calix[4]arene) species as determined by a combination of analytical methods including X-ray crystallography. An exception is a zinc(II) dithiocarbamate upper rim calix[4]arene assembly which is monomeric in nature. Electrochemical investigations reveal the bimetallic copper(II) bis(calix[4]arene) systems can electrochemically sense dihydrogen phosphate and carboxylate anions via significant cathodic perturbations of the respective copper(II)/(III) dithiocarbamate oxidation wave.

Detailed glycan analysis of serum glycoproteins of patients with congenital disorders of glycosylation indicates the specific defective glycan processing step and provides an insight into pathogenesis.

The fundamental importance of correct protein glycosylation is abundantly clear in a group of diseases known as congenital disorders of glycosylation (CDGs). In these diseases, many biological functions are compromised, giving rise to a wide range of severe clinical conditions. By performing detailed analyses of the total serum glycoproteins as well as isolated transferrin and IgG, we have directly correlated aberrant glycosylation with a faulty glycosylation processing step.