Defining variant-resistant epitopes targeted by SARS-CoV-2 antibodies: A global consortium study.

Antibody-based therapeutics and vaccines are essential to combat COVID-19 morbidity and mortality after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Multiple mutations in SARS-CoV-2 that could impair antibody defenses propagated in human-to-human transmission and spillover or spillback events between humans and animals. To develop prevention and therapeutic strategies, we formed an international consortium to map the epitope landscape on the SARS-CoV-2 spike protein, defining and structurally illustrating seven receptor binding domain (RBD)­directed antibody communities with distinct footprints and competition profiles. Pseudovirion-based neutralization assays reveal spike mutations, individually and clustered together in variants, that affect antibody function among the communities. Key classes of RBD-targeted antibodies maintain neutralization activity against these emerging SARS-CoV-2 variants. These results provide a framework for selecting antibody treatment cocktails and understanding how viral variants might affect antibody therapeutic efficacy.

Biologically Active Ultra-Simple Proteins Reveal Principles of Transmembrane Domain Interactions.

Specific interactions between the helical membrane-spanning domains of transmembrane proteins play central roles in the proper folding and oligomerization of these proteins. However, the relationship between the hydrophobic amino acid sequences of transmembrane domains and their functional interactions is in most cases unknown. Here, we use ultra-simple artificial proteins to systematically study the sequence basis for transmembrane domain interactions. We show that most short homopolymeric polyleucine transmembrane proteins containing single amino acid substitutions can activate the platelet-derived growth factor ß receptor or the erythropoietin receptor in cultured mouse cells, resulting in cell transformation or proliferation. These proteins displayed complex patterns of activity that were markedly affected by seemingly minor sequence differences in the ultra-simple protein itself or in the transmembrane domain of the target receptor, and the effects of these sequence differences are not additive. In addition, specific leucine residues along the length of these proteins are required for activity, and the positions of these required leucines differ based on the identity and position of the central substituted amino acid. Our results suggest that these ultra-simple proteins use a variety of molecular mechanisms to activate the same target and that diversification of transmembrane domain sequences over the course of evolution minimized off-target interactions.

Transmembrane Protein Aptamer Induces Cooperative Signaling by the EPO Receptor and the Cytokine Receptor ß-Common Subunit.

The erythropoietin receptor (EPOR) plays an essential role in erythropoiesis and other cellular processes by forming distinct signaling complexes composed of EPOR homodimers or hetero-oligomers between the EPOR and another receptor, but the mechanism of heteroreceptor assembly and signaling is poorly understood. We report here a 46-residue, artificial transmembrane protein aptamer, designated ELI-3, that binds and activates the EPOR and induces growth factor independence in murine BaF3 cells expressing the EPOR. ELI-3 requires the transmembrane domain and JAK2-binding sites of the EPOR for activity, but not the cytoplasmic tyrosines that mediate canonical EPOR signaling. Instead, ELI-3-induced proliferation and activation of JAK/STAT signaling requires the transmembrane and cytoplasmic domains of the cytokine receptor ß-common subunit (ßcR) in addition to the EPOR. Moreover, ELI-3 fails to induce erythroid differentiation of primary human hematopoietic progenitor cells but inhibits nonhematopoietic cell death induced by serum withdrawal.

Biologically active LIL proteins built with minimal chemical diversity.

We have constructed 26-amino acid transmembrane proteins that specifically transform cells but consist of only two different amino acids. Most proteins are long polymers of amino acids with 20 or more chemically distinct side-chains. The artificial transmembrane proteins reported here are the simplest known proteins with specific biological activity, consisting solely of an initiating methionine followed by specific sequences of leucines and isoleucines, two hydrophobic amino acids that differ only by the position of a methyl group. We designate these proteins containing leucine (L) and isoleucine (I) as LIL proteins. These proteins functionally interact with the transmembrane domain of the platelet-derived growth factor ß-receptor and specifically activate the receptor to transform cells. Complete mutagenesis of these proteins identified individual amino acids required for activity, and a protein consisting solely of leucines, except for a single isoleucine at a particular position, transformed cells. These surprisingly simple proteins define the minimal chemical diversity sufficient to construct proteins with specific biological activity and change our view of what can constitute an active protein in a cellular context.

Understanding vaccines: a public imperative.

Though once a discovery greatly celebrated by the nation, the vaccine has come under fire in recent decades from skeptics, critics, and a movement set into motion by fraudulent scientists and fueled by frustrated parents looking for answers to the autism conundrum. There is enough denialist resistance to vaccination to bring upon renewed fear of young children and infants becoming infected with diseases, the threats of which had been functionally eradicated from the United States. In more recent years, the surge in independent online journalism and blogging has invited many to rapidly share their opinions with millions of readers and, importantly, has appeared to open the door for opinion to be portrayed as fact. As a result, many parents are inundated with horror stories of vaccine dangers, all designed to eat away at them emotionally while the medical and scientific communities have mounted their characteristic response by sharing the facts, the data, and all of the reliable peer-reviewed and well-cited research to show that vaccines are safe and effective. It has become clear to me that facts are no match for emotion, but perhaps an understanding behind vaccine methodology will help parents overcome these fears of vaccinating. By helping those who doubt vaccines better understand what vaccines really are and how they work in such an incredibly engineered fashion, we may have a stronger weapon than we realize in battling the emotional arsenal that comes from the fear and skepticism of vaccinating.

Orm proteins integrate multiple signals to maintain sphingolipid homeostasis.

Sphingolipids are structural components of membranes, and sphingolipid metabolites serve as signaling molecules. The first and rate-limiting step in sphingolipid synthesis is catalyzed by serine palmitoyltransferase (SPT). The recently discovered SPT-associated proteins, Orm1 and Orm2, are critical regulators of sphingolipids. Orm protein phosphorylation mediating feedback regulation of SPT activity occurs in response to multiple sphingolipid intermediates, including long chain base and complex sphingolipids. Both branches of the TOR signaling network, TORC1 and TORC2, participate in regulating sphingolipid synthesis via Orm phosphorylation in response to sphingolipid intermediates as well as nutritional conditions. Moreover, sphingolipid synthesis is regulated in response to endoplasmic reticulum (ER) stress by activation of a calcium- and calcineurin-dependent pathway via transcriptional induction of ORM2. Conversely, the calcium- and calcineurin-dependent pathway signals ER stress response upon lipid dysregulation in the absence of the Orm proteins to restore ER homeostasis.