Barry Honig

Monocytes comprise two major subsets, Ly6C hi classical monocytes and Ly6C lo nonclassical monocytes. Notch2 signaling in Ly6C hi monocytes triggers transition to Ly6C lo monocytes, which require Nr4a1, Bcl6, Irf2, and Cebpb. By comparison, less is known...

Self-recognition is a fundamental cellular process across evolution and forms the basis of neuronal self-avoidance1-4. Clustered protocadherins (Pcdh), comprising a large family of isoform-specific homophilic recognition molecules, play a pivotal role in neuronal self-avoidance required for mammalian brain development5-7. The probabilistic expression of different Pcdh isoforms confers unique identities upon neurons and forms the basis for neuronal processes to discriminate between self and non-self5, 6, 8. Whether this self-recognition mechanism exists in astrocytes, the other predominant cell type of the brain, remains unknown. Here, we report that a specific isoform in the Pcdhγ cluster, γC3, is highly enriched in human and murine astrocytes. Through genetic manipulation, we demonstrate that γC3 acts autonomously to regulate astrocyte morphogenesis in the mouse visual cortex. To determine if γC3 proteins act by promoting recognition between processes of the same astrocyte, we generated pairs of γC3 chimeric proteins capable of heterophilic binding to each other, but incapable of homophilic binding. Co-expressing complementary heterophilic binding isoform pairs in the same γC3 null astrocyte restored normal morphology. By contrast, chimeric γC3 proteins individually expressed in single γC3 null mutant astrocytes did not. These data establish that self-recognition is essential for astrocyte development in the mammalian brain and that, by contrast to neuronal self-recognition, a single Pcdh isoform is both necessary and sufficient for this process.

Computational free energy-based methods have the potential to significantly improve throughput and decrease costs of protein design efforts. Such methods must reach a high level of reliability, accuracy, and automation to be effectively deployed in practical industrial settings in a way that impacts protein design projects. Here, we present a benchmark study for the calculation of relative changes in protein-protein binding affinity for single point mutations across a variety of systems from the literature, using free energy perturbation (FEP+) calculations. We describe a method for robust treatment of alternate protonation states for titratable amino acids, which yields improved correlation with and reduced error compared to experimental binding free energies. Following careful analysis of the largest outlier cases in our dataset, we assess limitations of the default FEP+ protocols and introduce an automated script which identifies probable outlier cases that may require additional scrutiny and calculates an empirical correction for a subset of charge-related outliers. Through a series of three additional case study systems, we discuss how protein FEP+ can be applied to real-world protein design projects, and suggest areas of further study.

In Drosophila, two interacting adhesion protein families, Dprs and DIPs, coordinate the assembly of neural networks. While intercellular DIP/Dpr interactions have been well characterized, DIPs and Dprs are often co-expressed within the same cells, raising the question as to whether they also interact in cis. We show, in cultured cells and in vivo, that DIP-α and DIP-δ can interact in cis with their ligands, Dpr6/10 and Dpr12, respectively. When co-expressed in cis with their cognate partners, these Dprs regulate the extent of trans binding through competitive cis interactions. We demonstrate the neurodevelopmental effects of cis inhibition in fly motor neurons and in the mushroom body. We further show that a long disordered region of DIP-α at the C-terminus is required for cis but not trans interactions, likely because it alleviates geometric constraints on cis binding. Thus, the balance between cis and trans interactions plays a role in controlling neural development.

Although the gain of function (GOF) of p53 mutants is well recognized, it remains unclear whether different p53 mutants share the same cofactors to induce GOFs. In a proteomic screen, we identified BACH1 as a cellular factor that recognizes the p53 DNA-binding domain depending on its mutation status. BACH1 strongly interacts with p53R175H but fails to effectively bind wild-type p53 or other hotspot mutants in vivo for functional regulation. Notably, p53R175H acts as a repressor for ferroptosis by abrogating BACH1-mediated downregulation of SLC7A11 to enhance tumor growth; conversely, p53R175H promotes BACH1-dependent tumor metastasis by upregulating expression of pro-metastatic targets. Mechanistically, p53R175H-mediated bidirectional regulation of BACH1 function is dependent on its ability to recruit the histone demethylase LSD2 to target promoters and differentially modulate transcription …

E-cadherin plays a central role in cell-cell adhesion. The ectodomains of wild-type cadherins form a crystalline-like two-dimensional lattice in cell-cell interfaces mediated by both trans (apposed cell) and cis (same cell) interactions. In addition to these extracellular forces, adhesive strength is further regulated by cytosolic phenomena involving α and β catenin-mediated interactions between cadherin and the actin cytoskeleton. Cell-cell adhesion can be further strengthened under tension through mechanisms that have not been definitively characterized in molecular detail. Here we quantitatively determine the role of the cadherin ectodomain in mechanosensing. To this end, we devise an E-cadherin-coated emulsion system, in which droplet surface tension is balanced by protein binding strength to give rise to stable areas of adhesion. To reach the honeycomb/cohesive limit, an initial emulsion compression by …

We describe the Predicting Protein–Compound Interactions (PrePCI) database which comprises over 5 billion predicted interactions between 6.8 million chemical compounds and 19,797 human proteins. PrePCI relies on a proteome‐wide database of structural models based on both traditional modeling techniques and the AlphaFold Protein Structure Database. Sequence‐ and structural similarity‐based metrics are established between template proteins, T, in the Protein Data Bank that bind compounds, C, and query proteins in the model database, Q. When the metrics exceed threshold values, it is assumed that C also binds to Q with a likelihood ratio (LR) derived from machine learning. If the relationship is based on structural similarity, the LR is based on a scoring function that measures the extent to which C is compatible with the binding site of Q as described in the LT‐scanner algorithm. For every predicted …

The strength of binding between human angiotensin converting enzyme 2 (ACE2) and the receptor binding domain (RBD) of viral spike protein plays a role in the transmissibility of the SARS-CoV-2 virus. In this study we focus on a subset of RBD mutations that have been frequently observed in infected individuals and probe binding affinity changes to ACE2 using surface plasmon resonance (SPR) measurements and free energy perturbation (FEP) calculations. Our SPR results are largely in accord with previous studies but discrepancies do arise due to differences in experimental methods and to protocol differences even when a single method is used. Overall, we find that FEP performance is superior to that of other computational approaches examined as determined by agreement with experiment and, in particular, by its ability to identify stabilizing mutations. Moreover, the calculations successfully predict the …