Genetic analysis of mammalian color variation has provided fundamental insight into human biology and disease. In most vertebrates, two key genes, Agouti and Melanocortin 1 receptor (Mc1r), encode a ligand-receptor system that controls pigment type-switching, but in domestic dogs, a third gene is implicated, the K locus, whose genetic characteristics predict a previously unrecognized component of the melanocortin pathway. We identify the K locus as β-defensin 103 (CBD103) and show that its protein product binds with high affinity to the Mc1r and has a simple and strong effect on pigment type-switching in domestic dogs and transgenic mice. These results expand the functional role of β-defensins, a protein family previously implicated in innate immunity, and identify an additional class of ligands for signaling through melanocortin receptors. AUTHORS' SUMMARYThe marked spectrum of color and diversity of patterns that we see in mammals arises, unexpectedly, from variation in the quantity, quality, and regional distribution of just two types of pigment-black eumelanin and yellow pheomelanin. The appeal of unusual coat colors and patterns has motivated their selection in domestic animals, providing geneticists with a model for studying gene action and interaction that began a century ago and continues today. Most of the work has been carried out in laboratory mice, where studies of more than 100 different coat-color mutations have provided insight into stem cell biology (hair graying), biogenesis of intracellular organelles (pigmentary dilution), and hormonereceptor interactions (switching between the synthesis of eumelanin and pheomelanin). ‡ To whom correspondence should be addressed. gbarsh@stanford.ed. * These authors contributed equally to the work. The latter process-commonly known as pigment "type-switching"-is controlled primarily by the melanocortin system, in which a family of G protein-coupled receptors (identified by virtue of their response to α-melanocyte-stimulating hormone or adrenocorticotrophic hormone) has been implicated not only in pigmentation but also in cortisol production, body weight regulation, and exocrine gland secretion. In most mammals, pigment type-switching is controlled by two genes, the Melanocortin 1 receptor (Mc1r) and Agouti, which encode a seven transmembrane-domain receptor and its extracellular ligand, respectively. Indeed, our current understanding of melanocortin biology stems from the identification in laboratory mice of Mc1r mutations as the cause of recessive yellow and Agouti mutations as the cause of lethal yellow.Clarence Cook Little, who developed many of the original laboratory mouse strains and founded The Jackson Laboratory, was also one of the first dog geneticists. He recognized that dominant inheritance of a black coat was mediated differently in dogs than in other animals (1). Using classical linkage analysis, we realized that the dominant black gene represented a previously unrecognized component of the melanocortin pathway (2). Unexpectedly, we found ...

An essential mechanism for severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection begins with the viral spike protein binding to the human receptor protein angiotensin-converting enzyme II (ACE2). Here, we describe a stepwise engineering approach to generate a set of affinity optimized, enzymatically inactivated ACE2 variants that potently block SARS-CoV-2 infection of cells. These optimized receptor traps tightly bind the receptor binding domain (RBD) of the viral spike protein and prevent entry into host cells. We first computationally designed the ACE2–RBD interface using a two-stage flexible protein backbone design process that improved affinity for the RBD by up to 12-fold. These designed receptor variants were affinity matured an additional 14-fold by random mutagenesis and selection using yeast surface display. The highest-affinity variant contained seven amino acid changes and bound to the RBD 170-fold more tightly than wild-type ACE2. With the addition of the natural ACE2 collectrin domain and fusion to a human immunoglobulin crystallizable fragment (Fc) domain for increased stabilization and avidity, the most optimal ACE2 receptor traps neutralized SARS-CoV-2–pseudotyped lentivirus and authentic SARS-CoV-2 virus with half-maximal inhibitory concentrations (IC50s) in the 10- to 100-ng/mL range. Engineered ACE2 receptor traps offer a promising route to fighting infections by SARS-CoV-2 and other ACE2-using coronaviruses, with the key advantage that viral resistance would also likely impair viral entry. Moreover, such traps can be predesigned for viruses with known entry receptors for faster therapeutic response without the need for neutralizing antibodies isolated from convalescent patients.

An essential mechanism for SARS-CoV-1 and -2 infection begins with the viral spike protein binding to the human receptor protein angiotensin-converting enzyme II (ACE2). Here we describe a stepwise engineering approach to generate a set of affinity optimized, enzymatically inactivated ACE2 variants that potently block SARS-CoV-2 infection of cells. These optimized receptor traps tightly bind the receptor binding domain (RBD) of the viral spike protein and prevent entry into host cells. We first computationally designed the ACE2-RBD interface using a two-stage flexible protein backbone design process that improved affinity for the RBD by up to 12-fold. These designed receptor variants were affinity matured an additional 14-fold by random mutagenesis and selection using yeast surface display. The highest affinity variant contained seven amino acid changes and bound to the RBD 170-fold more tightly than wild-type ACE2. With the addition of the natural ACE2 collectrin domain and fusion to a human Fc domain for increased stabilization and avidity, the most optimal ACE2 receptor traps neutralized SARS-CoV-2 pseudotyped lentivirus and authentic SARS-CoV-2 virus with half-maximal inhibitory concentrations (IC50) in the tens of ng/ml range. Engineered ACE2 receptor traps offer a promising route to fighting infections by SARS-CoV-2 and other ACE2-utilizing coronaviruses, with the key advantage that viral resistance would also likely impair viral entry. Moreover, such traps can be pre-designed for viruses with known entry receptors for faster therapeutic response without the need for neutralizing antibodies isolated or generated from convalescent patients.

β-Defensins are cationic host defense peptides that form an amphipathic structure stabilized by three intramolecular disulfide bonds. They are key players in innate and adaptive immunity and have recently been shown to limit the production of pro-inflammatory cytokines in TLR4-stimulated macrophages. In the present study, we investigate the mechanism underlying the anti-inflammatory effect of human β-defensin 3 (hBD3). We show that the canonical structure of hBD3 is required for this immunosuppressive effect and that hBD3 rapidly associates with and enters macrophages. Examination of the global effect of hBD3 on transcription in TLR4-stimulated macrophages shows that hBD3 inhibits the transcription of pro-inflammatory genes. Among the altered genes there is significant enrichment of groups involved in the positive regulation of NF-κB including components of Toll-like receptor signaling pathways. We confirm these observations by showing corresponding decreases in protein levels of pro-inflammatory cytokines and cell surface molecules. In addition, we show that hBD3 reduces NF-κB signaling in cells transfected with MyD88 or TRIF and that hBD3 inhibits the TLR4 response in both MyD88- and TRIF-deficient macrophages. Taken together these findings suggest that the mechanism of hBD3 anti-inflammatory activity involves specific targeting of TLR signaling pathways resulting in transcriptional repression of pro-inflammatory genes.

The melanocortin 1 receptor (MC1R), a Gs protein-coupled receptor, plays an important role in human pigmentation. We investigated the regulation of expression and activity of the MC1R in primary human melanocyte cultures. Human beta defensin 3 (HBD3) acted as an antagonist for MC1R, inhibiting the α-melanocortin (α-MSH)-induced increase in the activities of adenylate cyclase, and tyrosinase, the rate-limiting enzyme for melanogenesis. α-Melanocortin and forskolin, which activate adenylate cyclase, and 12-o-tetradecanoyl phorbol 13-acetate, which activates PKC, increased, while exposure to ultraviolet radiation (UV) reduced, MC1R gene and membrane protein expression. Brief treatment with α-MSH resulted in MC1R desensitization, while continuous treatment up to 3 hours caused a steady rise in cAMP, suggesting receptor recycling. Pretreatment with agouti signaling protein or HBD3 prohibited responsiveness to α-MSH, but not forskolin, suggesting receptor desensitization by these antagonists. Melanocytes from different donors expressed different levels of the G-protein-coupled receptor kinases (GRK) 2, 3, 5, and 6, and β-arrestin 1. Therefore, in addition to MC1R genotype, regulation of MC1R expression and activity is expected to affect human pigmentation and the responses to UV.

Ifitm3 was previously identified as an endosomal protein that blocks viral infection 1 – 3 . Studying clinical cohorts of B-cell leukemia and lymphoma patients, we identified IFITM3 as a strong predictor of poor outcome. In normal resting B-cells, Ifitm3 was minimally expressed and mainly localized in endosomes. However, B-cell receptor (BCR) engagement induced expression of Ifitm3 and phosphorylation at Y20, resulting in accumulation at the cell surface. In B-cell leukemia, oncogenic kinases phosphorylate IFITM3-Y20, causing constitutive plasma membrane localization. Ifitm3 ˉ / ˉ naïve B-cells developed at normal numbers; however, germinal center formation and production of antigen-specific antibodies were compromised. Oncogenes that induce development of leukemia and lymphoma failed to transform Ifitm3 ˉ / ˉ B-cells. Conversely, the phospho-mimetic IFITM3-Y20E induced oncogenic PI3K-signaling and initiated transformation of pre-malignant B-cells. Mechanistic experiments revealed that Ifitm3 functions as PIP3-scaffold and central amplifier of PI3K signaling. PI3K signal-amplification depends on Ifitm3 scaffolding PIP3-accumulation via two lysine residues (K83 and K104) in its conserved intracellular loop. In Ifitm3 ˉ / ˉ B-cells, lipid rafts were depleted of PIP3, resulting in defective expression of >60 lipid raft-associated surface receptors, impaired BCR-signaling and cellular adhesion. We conclude that phosphorylation of IFITM3 upon B-cell antigen-encounter induces a dynamic switch from antiviral effector functions in endosomes to a PI3K-amplification loop at the cell surface. IFITM3-dependent amplification of PI3K-signaling in part downstream of the BCR is critical to enable rapid expansion of B-cells with high affinity to antigen. In addition, multiple oncogenes depend on IFITM3 to assemble PIP3-dependent signaling complexes and amplify PI3K-signaling for malignant transformation.

Summary The β-defensins are a class of small, cationic proteins first recognized as antimicrobial components of the innate and adaptive immune system. More recently, one of the major β-defensins produced in skin, β-defensin 3, has been discovered to function as a melanocortin receptor ligand in vivo and in vitro, but its biophysical and pharmacological basis of action has been enigmatic. Here we report functional and biochemical studies focused on human β-defensin 3 (HBD3) and melanocortin receptors 1 and 4. Genetic and pharmacologic studies indicate that HBD3 acts as a neutral melanocortin receptor antagonist, capable of blocking the action of either stimulatory agonists, such as α-melanocyte stimulating hormone, or inhibitory inverse agonists such as Agouti signaling protein (Asip) and Agouti-related protein (Agrp). A comprehensive structure-function analysis demonstrates that two patches of positively charged residues, located on opposite poles of HBD3 and spatially organized by the compact β-defensin fold, are primarily responsible for high affinity binding to melanocortin receptors. These findings identify a distinct mode of melanocortin receptor-ligand interactions based primarily on electrostatic complementarity, with implications for designing ligands that target melanocortin and potentially other seven transmembrane receptors.

Malignant transformation typically involves multiple genetic lesions whose combined activity gives rise to cancer 1 . Our analysis of 1,148 patient-derived B-cell leukemia (B-ALL) samples revealed that individual mutations did not promote leukemogenesis unless they converged on one single oncogenic pathway characteristic for the differentiation stage of transformed B cells. Mutations not aligned with the central oncogenic driver activated divergent pathways and subverted transformation. Oncogenic lesions in B-ALL frequently mimic cytokine receptor signaling at the pro-B cell stage (through activation of STAT5) 2 – 4 or the pre-B cell receptor in more mature cells (through activation of ERK) 5 – 8 . STAT5- and ERK-activating lesions were frequently found but only co-occurred in ~3% of cases ( P =2.2E-16). Single-cell mutation and phosphoprotein analyses revealed the segregation of oncogenic STAT5- or ERK-activation to competing clones. STAT5 and ERK engaged opposing biochemical and transcriptional programs orchestrated by MYC and BCL6, respectively. Genetic reactivation of the divergent (suppressed) pathway came at the expense of the principal oncogenic driver and reversed transformation. Conversely, deletion of divergent pathway components accelerated leukemogenesis. Thus, persistence of divergent signaling pathways represents a powerful barrier to transformation while convergence on one principal driver defines a central event in leukemia-initiation. Pharmacological reactivation of suppressed divergent circuits strongly synergized with inhibition of the principal oncogenic driver. Hence, reactivation of divergent pathways can be leveraged as a previously unrecognized strategy to deepen treatment responses.