Deep mutational scanning of proteins in mammalian cells.

Protein mutagenesis is essential for unveiling the molecular mechanisms underlying protein function in health, disease, and evolution. In the past decade, deep mutational scanning methods have evolved to support the functional analysis of nearly all possible single-amino acid changes in a protein of interest. While historically these methods were developed in lower organisms such as E.

The Biologist's Guide to the Glucocorticoid Receptor's Structure.

The glucocorticoid receptor α (GRα) is a member of the nuclear receptor superfamily and functions as a glucocorticoid (GC)-responsive transcription factor. GR can halt inflammation and kill off cancer cells, thus explaining the widespread use of glucocorticoids in the clinic. However, side effects and therapy resistance limit GR's therapeutic potential, emphasizing the importance of resolving all of GR's context-specific action mechanisms.

Selective IL-1 activity on CD8 T cells empowers antitumor immunity and synergizes with neovasculature-targeted TNF for full tumor eradication.

Background: Clinical success of therapeutic cancer vaccines depends on the ability to mount strong and durable antitumor T cell responses. To achieve this, potent cellular adjuvants are highly needed. Interleukin-1β (IL-1β) acts on CD8 T cells and promotes their expansion and effector differentiation, but toxicity and undesired tumor-promoting side effects hamper efficient clinical application of this cytokine.

An affinity-enhanced, broadly neutralizing heavy chain-only antibody protects against SARS-CoV-2 infection in animal models.

Broadly neutralizing antibodies are an important treatment for individuals with coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Antibody-based therapeutics are also essential for pandemic preparedness against future outbreaks. Camelid-derived single domain antibodies (VHHs) exhibit potent antimicrobial activity and are being developed as SARS-CoV-2–neutralizing antibody-like therapeutics.

Specific targeting of IL-1β activity to CD8 T cells allows for safe use as a vaccine adjuvant.

Annual administration and reformulation of influenza vaccines is required for protection against seasonal infections. However, the induction of strong and long-lasting T cells is critical to reach broad and potentially lifelong antiviral immunity. The NLRP3 inflammasome and its product interleukin-1β (IL-1β) are pivotal mediators of cellular immune responses to influenza, yet, overactivation of these systems leads to side effects, which hamper clinical applications.

Safe eradication of large established tumors using neovasculature-targeted tumor necrosis factor-based therapies.

Systemic toxicities have severely limited the clinical application of tumor necrosis factor (TNF) as an anticancer agent. Activity-on-Target cytokines (AcTakines) are a novel class of immunocytokines with improved therapeutic index. A TNF-based AcTakine targeted to CD13 enables selective activation of the tumor neovasculature without any detectable toxicity in vivo.

The Small GTPase Arf6: An Overview of Its Mechanisms of Action and of Its Role in Host⁻Pathogen Interactions and Innate Immunity.

The small GTase Arf6 has several important functions in intracellular vesicular trafficking and regulates the recycling of different types of cargo internalized via clathrin-dependent or -independent endocytosis. It activates the lipid modifying enzymes PIP 5-kinase and phospholipase D, promotes actin polymerization, and affects several functionally distinct processes in the cell. Arf6 is used for the phagocytosis of pathogens and can be directly or indirectly targeted by various pathogens to block phagocytosis or induce the uptake of intracellular pathogens.

Straightforward Protein-Protein Interaction Interface Mapping via Random Mutagenesis and Mammalian Protein Protein Interaction Trap (MAPPIT).

The MAPPIT (mammalian protein protein interaction trap) method allows high-throughput detection of protein interactions by very simple co-transfection of three plasmids in HEK293T cells, followed by a luciferase readout. MAPPIT detects a large percentage of all protein interactions, including those requiring posttranslational modifications and endogenous or exogenous ligands. Here, we present a straightforward method that allows detailed mapping of interaction interfaces via MAPPIT.

The N-terminal p.(Ser38Cys) TIMP3 mutation underlying Sorsby fundus dystrophy is a founder mutation disrupting an intramolecular disulfide bond.

Sorsby fundus dystrophy (SFD) is a macular degeneration caused by mutations in TIMP3, the majority of which introduce a novel cysteine. However, the exact molecular mechanisms underlying SFD remain unknown. We aimed to provide novel insights into the functional consequences of a distinct N-terminal mutation.

Correction: Biallelic sequence and structural variants in RAX2 are a novel cause for autosomal recessive inherited retinal disease.

The original version of this Article contained an incorrect version of Fig. 3, which included two variants initially shown in black text in Fig. 3a that the authors removed from the final manuscript.

Biallelic sequence and structural variants in RAX2 are a novel cause for autosomal recessive inherited retinal disease.

Purpose: RAX2 encodes a homeobox-containing transcription factor, in which four monoallelic pathogenic variants have been described in autosomal dominant cone-dominated retinal disease.

Methods: Exome sequencing in a European cohort with inherited retinal disease (IRD) (n = 2086) was combined with protein structure modeling of RAX2 missense variants, bioinformatics analysis of deletion breakpoints, haplotyping of RAX2 variant c.335dup, and clinical assessment of biallelic RAX2-positive cases and carrier family members.

Author Correction: Virtual screening for inhibitors of the human TSLP:TSLPR interaction.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Biallelic and monoallelic ESR2 variants associated with 46,XY disorders of sex development.

Purpose: Disorders or differences of sex development (DSDs) are rare congenital conditions characterized by atypical sex development. Despite advances in genomic technologies, the molecular cause remains unknown in 50% of cases.

Methods: Homozygosity mapping and whole-exome sequencing revealed an ESR2 variant in an individual with syndromic 46,XY DSD.

Virtual screening for inhibitors of the human TSLP:TSLPR interaction.

The pro-inflammatory cytokine thymic stromal lymphopoietin (TSLP) plays a pivotal role in the pathophysiology of various allergy disorders that are mediated by type 2 helper T cell (Th2) responses, such as asthma and atopic dermatitis. TSLP forms a ternary complex with the TSLP receptor (TSLPR) and the interleukin-7-receptor subunit alpha (IL-7Rα), thereby activating a signaling cascade that culminates in the release of pro-inflammatory mediators. In this study, we conducted an in silico characterization of the TSLP:TSLPR complex to investigate the drugability of this complex.

TYK2-induced phosphorylation of Y640 suppresses STAT3 transcriptional activity.

STAT3 is a pleiotropic transcription factor involved in homeostatic and host defense processes in the human body. It is activated by numerous cytokines and growth factors and generates a series of cellular effects. Of the STAT-mediated signal transduction pathways, STAT3 transcriptional control is best understood.

RNF41 interacts with the VPS52 subunit of the GARP and EARP complexes.

RNF41 (Ring Finger Protein 41) is an E3 ubiquitin ligase involved in the intracellular sorting and function of a diverse set of substrates. Next to BRUCE and Parkin, RNF41 can directly ubiquitinate ErbB3, IL-3, EPO and RARα receptors or downstream signaling molecules such as Myd88, TBK1 and USP8. In this way it can regulate receptor signaling and routing.

Structure and antagonism of the receptor complex mediated by human TSLP in allergy and asthma.

The pro-inflammatory cytokine thymic stromal lymphopoietin (TSLP) is pivotal to the pathophysiology of widespread allergic diseases mediated by type 2 helper T cell (Th2) responses, including asthma and atopic dermatitis. The emergence of human TSLP as a clinical target against asthma calls for maximally harnessing its therapeutic potential via structural and mechanistic considerations. Here we employ an integrative experimental approach focusing on productive and antagonized TSLP complexes and free cytokine.

NR5A1 is a novel disease gene for 46,XX testicular and ovotesticular disorders of sex development.

Purpose: We aimed to identify the genetic cause in a cohort of 11 unrelated cases and two sisters with 46,XX SRY-negative (ovo)testicular disorders of sex development (DSD).

Methods: Whole-exome sequencing (n = 9), targeted resequencing (n = 4), and haplotyping were performed. Immunohistochemistry of sex-specific markers was performed on patients' gonads.

Isolated and Syndromic Retinal Dystrophy Caused by Biallelic Mutations in RCBTB1, a Gene Implicated in Ubiquitination.

Inherited retinal dystrophies (iRDs) are a group of genetically and clinically heterogeneous conditions resulting from mutations in over 250 genes. Here, homozygosity mapping and whole-exome sequencing (WES) in a consanguineous family revealed a homozygous missense mutation, c.973C>T (p.

Reconstructing the TIR Side of the Myddosome: a Paradigm for TIR-TIR Interactions.

Members of the Toll-like receptor and interleukin-1 (IL-1) receptor families all signal via Toll/IL-1R (TIR) domain-driven assemblies with adaptors such as MyD88. We here combine the mammalian two-hybrid system MAPPIT and saturation mutagenesis to complement and extend crystallographic and nuclear magnetic resonance data, and reveal how TIR domains interact. We fully delineate the interaction sites on the MyD88 TIR domain for homo-oligomerization and for interaction with Mal and TLR4.

Leptin: From structural insights to the design of antagonists.

After its discovery in 1994, it soon became clear that leptin acts as an adipocyte-derived hormone with a central role in the control of body weight and energy homeostasis. However, a growing body of evidence has revealed that leptin is a pleiotropic cytokine with activities on many peripheral cell types. Inappropriate leptin signaling can promote autoimmunity, certain cardiovascular diseases, elevated blood pressure and cancer, which makes leptin and the leptin receptor interesting targets for antagonism.