Affinity-matured DLL4 ligands as broad-spectrum modulators of Notch signaling.

The Notch pathway regulates cell fate decisions and is an emerging target for regenerative and cancer therapies. Recombinant Notch ligands are attractive candidates for modulating Notch signaling; however, their intrinsically low receptor-binding affinity restricts their utility in biomedical applications. To overcome this limitation, we evolved variants of the ligand Delta-like 4 with enhanced affinity and cross-reactivity. A consensus variant with maximized binding affinity, DeltaMAX, binds human and murine Notch receptors with 500- to 1,000-fold increased affinity compared with wild-type human Delta-like 4. DeltaMAX also potently activates Notch in plate-bound, bead-bound and cellular formats. When administered as a soluble decoy, DeltaMAX inhibits Notch in reporter and neuronal differentiation assays, highlighting its dual utility as an agonist or antagonist. Finally, we demonstrate that DeltaMAX stimulates increased proliferation and expression of effector mediators in T cells. Taken together, our data define DeltaMAX as a versatile tool for broad-spectrum activation or inhibition of Notch signaling.

Structural basis for mouse LAG3 interactions with the MHC class II molecule I-Ab.

The immune checkpoint protein, Lymphocyte activation gene-3 (LAG3), binds Major Histocompatibility Complex Class II (MHC-II) and suppresses T cell activation. Despite the recent FDA approval of a LAG3 inhibitor for the treatment of melanoma, how LAG3 engages MHC-II on the cell surface remains poorly understood. Here, we determine the 3.84 Å-resolution structure of mouse LAG3 bound to the MHC-II molecule I-Ab, revealing that domain 1 (D1) of LAG3 binds a conserved, membrane-proximal region of MHC-II spanning both the α2 and ß2 subdomains. LAG3 dimerization restricts the intermolecular spacing of MHC-II molecules, which may attenuate T cell activation by enforcing suboptimal signaling geometry. The LAG3-MHC-II interface overlaps with the MHC-II-binding site of the T cell coreceptor CD4, implicating disruption of CD4-MHC-II interactions as a mechanism for LAG3 immunosuppressive function. Lastly, antibody epitope analysis indicates that multiple LAG3 inhibitors do not recognize the MHC-II-binding interface of LAG3, suggesting a role for functionally distinct mechanisms of LAG3 antagonism in therapeutic development.

Engineering synthetic agonists for targeted activation of Notch signaling.

Notch signaling regulates cell fate decisions and has context-dependent tumorigenic or tumor suppressor functions. Although there are several classes of Notch inhibitors, the mechanical force requirement for Notch receptor activation has hindered attempts to generate soluble agonists. To address this problem, we engineered synthetic Notch agonist (SNAG) proteins by tethering affinity-matured Notch ligands to antibodies or cytokines that internalize their targets. This bispecific format enables SNAGs to "pull" on mechanosensitive Notch receptors, triggering their activation in the presence of a desired biomarker. We successfully developed SNAGs targeting six independent surface markers, including the tumor antigens PDL1, CD19, and HER2, and the immunostimulatory receptor CD40. SNAGs targeting CD40 increase expansion of central memory γδ T cells from peripheral blood, highlighting their potential to improve the phenotype and yield of low-abundance T cell subsets. These insights have broad implications for the pharmacological activation of mechanoreceptors and will expand our ability to modulate Notch signaling in biotechnology.

New tricks for an old pathway: emerging Notch-based biotechnologies and therapeutics.

The Notch pathway regulates a diverse array of cell fate decisions, making it an enticing target in cancer therapy and regenerative medicine. During the early stages of Notch drug development, off-target toxicity precluded the approval of Notch inhibitors for the treatment of cancer. However, recent advances in our understanding of Notch structure and signaling have led to the development of several innovative Notch-based biotechnologies. In addition to new classes of inhibitors, pharmacological Notch activators have been shown to enhance osteogenesis and various aspects of T cell function. Furthermore, the mechanosensitive negative regulatory region (NRR) of the Notch receptor has been converted into synthetic Notch (synNotch) receptors with fully customizable signaling circuits. We review emergent Notch-based compounds, biologics, and cell therapies while highlighting the challenges and opportunities they face on the path to clinical development.

Structure of the planar cell polarity cadherins Fat4 and Dachsous1.

The atypical cadherins Fat and Dachsous are key regulators of cell growth and animal development. In contrast to classical cadherins, which form homophilic interactions to segregate cells, Fat and Dachsous cadherins form heterophilic interactions to induce cell polarity within tissues. Here, we determine the co-crystal structure of the human homologs Fat4 and Dachsous1 (Dchs1) to establish the molecular basis for Fat-Dachsous interactions. The binding domains of Fat4 and Dchs1 form an extended interface along extracellular cadherin (EC) domains 1-4 of each protein. Biophysical measurements indicate that Fat4-Dchs1 affinity is among the highest reported for cadherin superfamily members, which is attributed to an extensive network of salt bridges not present in structurally similar protocadherin homodimers. Furthermore, modeling suggests that unusual extracellular phosphorylation modifications directly modulate Fat-Dachsous binding by introducing charged contacts across the interface. Collectively, our analyses reveal how the molecular architecture of Fat4-Dchs1 enables them to form long-range, high-affinity interactions to maintain planar cell polarity.

Fucosylated Proteome Profiling Identifies a Fucosylated, Non-Ribosomal, Stress-Responsive Species of Ribosomal Protein S3.

Alterations in genes encoding for proteins that control fucosylation are known to play causative roles in several developmental disorders, such as Dowling-Degos disease 2 and congenital disorder of glycosylation type IIc (CDGIIc). Recent studies have provided evidence that changes in fucosylation can contribute to the development and progression of several different types of cancers. It is therefore important to gain a detailed understanding of how fucosylation is altered in disease states so that interventions may be developed for therapeutic purposes. In this report, we find that fucosylation occurs on many intracellular proteins. This is an interesting finding, as the fucosylation machinery is restricted to the secretory pathway and is thought to predominately affect cell-membrane-bound and secreted proteins. We find that Ribosomal protein S3 (RPS3) is fucosylated in normal tissues and in cancer cells, and that the extent of its fucosylation appears to respond to stress, including MAPK inhibitors, suggesting a new role in posttranslational protein function. Our data identify a new ribosome-independent species of fucosylated RPS3 that interacts with proteins involved in posttranscriptional regulation of RNA, such as Heterogeneous nuclear ribonucleoprotein U (HNRNPU), as well as with a predominance of non-coding RNAs. These data highlight a novel role for RPS3, which, given previously reported oncogenic roles for RPS3, might represent functions that are perturbed in pathologies such as cancer. Together, our findings suggest a previously unrecognized role for fucosylation in directly influencing intracellular protein functions.