Biophysical mapping of TREM2-ligand interactions reveals shared surfaces for engagement of multiple Alzheimer's disease ligands.

TREM2 is a signaling receptor expressed on microglia that has emerged as an important drug target for Alzheimer's disease and other neurodegenerative diseases. While a number of TREM2 ligands have been identified, little is known regarding the structural details of how they engage. To better understand this, we created a protein library of 28 different TREM2 variants that could be used to map interactions with various ligands using biolayer interferometry.

Biophysical mapping of TREM2-ligand interactions reveals shared surfaces for engagement of multiple Alzheimer's disease ligands.

TREM2 is a signaling receptor expressed on microglia that has emerged as an important drug target for Alzheimer's disease and other neurodegenerative diseases. While a number of TREM2 ligands have been identified, little is known regarding the structural details of how they engage. To better understand this, we created a protein library of 28 different TREM2 variants that could be used to map interactions with various ligands using biolayer interferometry.

Modulation of TMEM16B channel activity by the calcium-activated chloride channel regulator 4 (CLCA4) in human cells.

The Ca-activated Cl channel regulator CLCA1 potentiates the activity of the Ca-activated Cl channel (CaCC) TMEM16A by directly engaging the channel at the cell surface, inhibiting its reinternalization and increasing Ca-dependent Cl current (I) density. We now present evidence of functional pairing between two other CLCA and TMEM16 protein family members, namely CLCA4 and the CaCC TMEM16B. Similar to CLCA1, (i) CLCA4 is a self-cleaving metalloprotease, and the N-terminal portion (N-CLCA4) is secreted; (ii) the von Willebrand factor type A (VWA) domain in N-CLCA4 is sufficient to potentiate I in HEK293T cells; and (iii) this is mediated by the metal ion-dependent adhesion site motif within VWA.

Production of Eukaryotic Glycoproteins for Structural and Functional Studies Using Expi293F Cells.

Milligram quantities of pure proteins are required for structural, functional, and pharmaceutical screening studies. These requirements can be challenging for a majority of important therapeutic targets that are secreted glycoproteins, receptors, membrane proteins, or large cytosolic complexes. Here, we present protocols for producing and purifying large amounts of secreted glycoproteins using the mammalian cell-based Expi293F system via large-scale transient transfection.

Lrp1 is a host entry factor for Rift Valley fever virus.

Rift Valley fever virus (RVFV) is a zoonotic pathogen with pandemic potential. RVFV entry is mediated by the viral glycoprotein (Gn), but host entry factors remain poorly defined. Our genome-wide CRISPR screen identified low-density lipoprotein receptor-related protein 1 (mouse Lrp1/human LRP1), heat shock protein (Grp94), and receptor-associated protein (RAP) as critical host factors for RVFV infection.

Functional insights from biophysical study of TREM2 interactions with apoE and Aβ.

Introduction: Triggering receptor expressed on myeloid cells-2 (TREM2) is an immune receptor expressed on microglia that also can become soluble (sTREM2). How TREM2 engages different ligands remains poorly understood.

Methods: We used comprehensive biolayer interferometry (BLI) analysis to investigate TREM2 and sTREM2 interactions with apolipoprotein E (apoE) and monomeric amyloid beta (Aβ) (mAβ42).

Structural and Biophysical Analysis of the CLCA1 VWA Domain Suggests Mode of TMEM16A Engagement.

The secreted protein calcium-activated chloride channel regulator 1 (CLCA1) utilizes a von Willebrand factor type A (VWA) domain to bind to and potentiate the calcium-activated chloride channel TMEM16A. To gain insight into this unique potentiation mechanism, we determined the 2.0-Å crystal structure of human CLCA1 VWA bound to Ca.

The Ebola Viral Protein 35 N-Terminus Is a Parallel Tetramer.

Members of Mononegavirales, the order that includes nonsegmented negative sense RNA viruses (NNSVs), encode a small number of multifunctional proteins. In members of the Filoviridae family, virus protein 35 (VP35) facilitates immune evasion and functions as an obligatory cofactor for viral RNA synthesis. VP35 functions in a manner orthologous to that of phosphoproteins from other NNSVs.

YbtT is a low-specificity type II thioesterase that maintains production of the metallophore yersiniabactin in pathogenic enterobacteria.

Clinical isolates of , , and frequently secrete the small molecule metallophore yersiniabactin (Ybt), which passivates and scavenges transition metals during human infections. YbtT is encoded within the Ybt biosynthetic operon and is critical for full Ybt production in bacteria. However, its biosynthetic function has been unclear because it is not essential for Ybt production by the reconstituted nonribosomal peptide synthetase/polyketide synthase (NRPS/PKS) pathway.

Limiting Respiratory Viral Infection by Targeting Antiviral and Immunological Functions of BST-2/Tetherin: Knowledge and Gaps.

Recent findings regarding the cellular biology and immunology of BST-2 (also known as tetherin) indicate that its function could be exploited as a universal replication inhibitor of enveloped respiratory viruses (e.g., influenza, respiratory syncytial virus, etc.

TREM2-Ligand Interactions in Health and Disease.

The protein triggering receptor expressed on myeloid cells-2 (TREM2) is an immunomodulatory receptor with a central role in myeloid cell activation and survival. In recent years, the importance of TREM2 has been highlighted by the identification of coding variants that increase risk for Alzheimer's disease and other neurodegenerative diseases. Animal studies have further shown the importance of TREM2 in neurodegenerative and other inflammatory disease models including chronic obstructive pulmonary disease, multiple sclerosis, and stroke.

Modulation of TMEM16A channel activity by the von Willebrand factor type A (VWA) domain of the calcium-activated chloride channel regulator 1 (CLCA1).

Calcium-activated chloride channels (CaCCs) are key players in transepithelial ion transport and fluid secretion, smooth muscle constriction, neuronal excitability, and cell proliferation. The CaCC regulator 1 (CLCA1) modulates the activity of the CaCC TMEM16A/Anoctamin 1 (ANO1) by directly engaging the channel at the cell surface, but the exact mechanism is unknown. Here we demonstrate that the von Willebrand factor type A (VWA) domain within the cleaved CLCA1 N-terminal fragment is necessary and sufficient for this interaction.

First comprehensive structural and biophysical analysis of MAPK13 inhibitors targeting DFG-in and DFG-out binding modes.

Background: P38 MAP kinases are centrally involved in mediating extracellular signaling in various diseases. While much attention has previously been focused on the ubiquitously expressed family member MAPK14 (p38α), recent studies indicate that family members such as MAPK13 (p38δ) display a more selective cellular and tissue expression and might therefore represent a specific kinase to target in certain diseases.

Methods: To facilitate the design of potent and specific inhibitors, we present here the structural, biophysical, and functional characterization of two new MAPK13-inhibitor complexes, as well as the first comprehensive structural, biophysical, and functional analysis of MAPK13 complexes with four different inhibitor compounds of greatly varying potency.

Data publication with the structural biology data grid supports live analysis.

Access to experimental X-ray diffraction image data is fundamental for validation and reproduction of macromolecular models and indispensable for development of structural biology processing methods. Here, we established a diffraction data publication and dissemination system, Structural Biology Data Grid (SBDG; data.sbgrid.

Novel Roles for Chloride Channels, Exchangers, and Regulators in Chronic Inflammatory Airway Diseases.

Chloride transport proteins play critical roles in inflammatory airway diseases, contributing to the detrimental aspects of mucus overproduction, mucus secretion, and airway constriction. However, they also play crucial roles in contributing to the innate immune properties of mucus and mucociliary clearance. In this review, we focus on the emerging novel roles for a chloride channel regulator (CLCA1), a calcium-activated chloride channel (TMEM16A), and two chloride exchangers (SLC26A4/pendrin and SLC26A9) in chronic inflammatory airway diseases.

PARP9-DTX3L ubiquitin ligase targets host histone H2BJ and viral 3C protease to enhance interferon signaling and control viral infection.

Enhancing the response to interferon could offer an immunological advantage to the host. In support of this concept, we used a modified form of the transcription factor STAT1 to achieve hyper-responsiveness to interferon without toxicity and markedly improve antiviral function in transgenic mice and transduced human cells. We found that the improvement depended on expression of a PARP9-DTX3L complex with distinct domains for interaction with STAT1 and for activity as an E3 ubiquitin ligase that acted on host histone H2BJ to promote interferon-stimulated gene expression and on viral 3C proteases to degrade these proteases via the immunoproteasome.

CLCA1 and TMEM16A: the link towards a potential cure for airway diseases.

The hallmark traits of chronic obstructive airway diseases are inflammation, airway constriction due to hyperreactivity and mucus overproduction. The current common treatments for asthma and chronic obstructive pulmonary disease target the first two traits with none currently targeting mucus overproduction. The main source of obstructive mucus production is mucus cell metaplasia (MCM), the transdifferentiation of airway epithelial cells into mucus-producing goblet cells, in the small airways.

TREM-2 promotes macrophage survival and lung disease after respiratory viral infection.

Viral infections and type 2 immune responses are thought to be critical for the development of chronic respiratory disease, but the link between these events needs to be better defined. Here, we study a mouse model in which infection with a mouse parainfluenza virus known as Sendai virus (SeV) leads to long-term activation of innate immune cells that drive IL-13-dependent lung disease. We find that chronic postviral disease (signified by formation of excess airway mucus and accumulation of M2-differentiating lung macrophages) requires macrophage expression of triggering receptor expressed on myeloid cells-2 (TREM-2).

The crystal structure of phosphorylated MAPK13 reveals common structural features and differences in p38 MAPK family activation.

The p38 MAP kinases (p38 MAPKs) represent an important family centrally involved in mediating extracellular signaling. Recent studies indicate that family members such as MAPK13 (p38δ) display a selective cellular and tissue expression and are therefore involved in specific diseases. Detailed structural studies of all p38 MAPK family members are crucial for the design of specific inhibitors.

Secreted CLCA1 modulates TMEM16A to activate Ca(2+)-dependent chloride currents in human cells.

Calcium-activated chloride channel regulator 1 (CLCA1) activates calcium-dependent chloride currents; neither the target, nor mechanism, is known. We demonstrate that secreted CLCA1 activates calcium-dependent chloride currents in HEK293T cells in a paracrine fashion, and endogenous TMEM16A/Anoctamin1 conducts the currents. Exposure to exogenous CLCA1 increases cell surface levels of TMEM16A and cellular binding experiments indicate CLCA1 engages TMEM16A on the surface of these cells.

Ebola virus VP24 targets a unique NLS binding site on karyopherin alpha 5 to selectively compete with nuclear import of phosphorylated STAT1.

During antiviral defense, interferon (IFN) signaling triggers nuclear transport of tyrosine-phosphorylated STAT1 (PY-STAT1), which occurs via a subset of karyopherin alpha (KPNA) nuclear transporters. Many viruses, including Ebola virus, actively antagonize STAT1 signaling to counteract the antiviral effects of IFN. Ebola virus VP24 protein (eVP24) binds KPNA to inhibit PY-STAT1 nuclear transport and render cells refractory to IFNs.

Preparation, crystallization, and preliminary crystallographic analysis of wild-type and mutant human TREM-2 ectodomains linked to neurodegenerative and inflammatory diseases.

TREM-2 (triggering receptor expressed on myeloid cells-2) is an innate immune receptor expressed on dendritic cells, macrophages, osteoclasts, and microglia. Recent genetic studies have reported the occurrence of point mutations in TREM-2 that correlate with a dramatically increased risk for the development of neurodegenerative diseases, including Alzheimer's disease, frontotemporal dementia, and Parkinson's disease. Structural and biophysical studies of wild-type and mutant TREM-2 ectodomains are required to understand the functional consequences of these mutations.

BST-2/tetherin: structural biology, viral antagonism, and immunobiology of a potent host antiviral factor.

BST-2 (also known as tetherin, CD317, or HM1.24) was first described as a potent interferon-inducible host antiviral factor nearly five years ago. Since that time, numerous reports have been published regarding the antiviral activity and immunological properties of this protein.

IL-13-induced airway mucus production is attenuated by MAPK13 inhibition.

Increased mucus production is a common cause of morbidity and mortality in inflammatory airway diseases, including asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis. However, the precise molecular mechanisms for pathogenic mucus production are largely undetermined. Accordingly, there are no specific and effective anti-mucus therapeutics.