SUMMARY Pellino proteins are RING E3 ubiquitin ligases involved in signaling events downstream of the Toll and interleukin-1 (IL-1) receptors, key initiators of innate immune and inflammatory responses. Pellino proteins associate with and ubiquitinate proteins in these pathways, including the interleukin-1 receptor associated kinase-1 (IRAK1). We determined the X-ray crystal structure of a Pellino2 fragment lacking only the RING domain. This structure reveals that the IRAK1-binding region of Pellino proteins consists largely of a previously unidentified forkhead-associated (FHA) domain. FHA domains are well-characterized phosphothreonine-binding modules, and this cryptic example in Pellino2 can drive interaction of this protein with phosphorylated IRAK1. The Pellino FHA domain is decorated with an unusual appendage or ‘wing’ composed of two long inserts that lie within the FHA homology region. Delineating how this E3 ligase associates with substrates, and how these interactions are regulated by phosphorylation, is crucial for a complete understanding of Toll/IL-1 receptor signaling.

The respiratory syncytial virus (RSV) fusion (F) glycoprotein is a major target of neutralizing antibodies arising from natural infection, and antibodies that specifically bind to the prefusion conformation of RSV F generally demonstrate the greatest neutralization potency. Prefusion-stabilized RSV F variants have been engineered as vaccine antigens, but crystal structures of these variants have revealed conformational differences in a key antigenic site located at the apex of the trimer, referred to as antigenic site Ø. Currently, it is unclear if flexibility in this region is an inherent property of prefusion RSV F or if it is related to inadequate stabilization of site Ø in the engineered variants. Therefore, we set out to investigate the conformational flexibility of antigenic site Ø, as well as the ability of the human immune system to recognize alternative conformations of this site, by determining crystal structures of prefusion RSV F bound to neutralizing human-derived antibodies AM22 and RSD5. Both antibodies bound with high affinity and were specific for the prefusion conformation of RSV F. Crystal structures of the complexes revealed that the antibodies recognized distinct conformations of antigenic site Ø, each diverging at a conserved proline residue located in the middle of an α-helix. These data suggest that antigenic site Ø exists as an ensemble of conformations, with individual antibodies recognizing discrete states. Collectively, these results have implications for the refolding of pneumovirus and paramyxovirus fusion proteins and should inform development of prefusion-stabilized RSV F vaccine candidates.

We have examined expression of L1 and the polysialic acid-associated form of the neural cell adhesion molecule (PSA-NCAM) in mouse embryos during the major period of axon growth in the retinofugal pathway to determine whether they are expressed in patterns that relate to the changes in axon organization in the pathway. Immunostaining for L1 and PSA-NCAM was found on all axons in the retina and the optic stalk. In the chiasm, while L1 immunoreactivity remained high on the axons, PSA-NCAM staining was obviously reduced. At the threshold of the optic tract, L1 immunoreactivity was maintained only in a subpopulation of axons, whereas PSA-NCAM staining was dramatically elevated in axons at the caudal part of the tract. Further investigations of the tract showed that both L1 and PSA-NCAM were preferentially expressed on the dorsal but not ventral optic axons, indicating a regionally specific change of both adhesion molecules on the axons at the chiasm-tract junction. Moreover, intense PSA-NCAM expression was also observed in the tract of postoptic commissure (TPOC), which lies immediately caudal to the optic tract. Immunohistochemical and retrograde tracing studies showed that these PSA-NCAM-positive axons arose from a population of cells rostral to the CD44-positive chiasmatic neurons. These findings indicate that, in addition to the chiasmatic neurons, these PSA-NCAM-positive diencephalic cells also contribute axons to the TPOC. These early generated commissural axons together with the regionally specific pattern of cell adhesion molecule expression on the optic axons may control formation of the partial retinotopic axon order in the optic tract through homophilic or heterophilic interactions that involve PSA-NCAM.

Elegant single molecule approaches have elucidated the mechanisms that cause RNA polymerase to pause during transcription. However, it is unclear whether these pausing events and their subsequent recovery occur within the cell as there are a large number of elongation factors that facilitate the progression of RNA polymerase (RNAP) through a chromatized genome. To explore the mechanisms of RNA polymerase elongation in vivo, we require experimental strategies that can observe transcription with the same resolution as can be obtained in vitro. Here we present an approach, native elongating transcript sequencing -NET-seq, that accomplishes this goal by exploiting the extraordinary stability of the DNA-RNA-RNAP ternary complex to capture nascent transcripts directly from live cells without crosslinking. The identity and abundance of the 3' end of purified transcripts are revealed by deep sequencing thus providing a quantitative measure of RNAP density with single nucleotide precision. Application of NET-seq in Saccharomyces cerevisiae reveals pervasive polymerase pausing and backtracking throughout the body of transcripts. Average pause density shows prominent peaks at each of the first four nucleosomes with the peak location occurring in good agreement with in vitro single molecule measurements. Thus nucleosome-induced pausing represents a major barrier to transcriptional elongation in vivo.

Upon stimulation of Toll‐like receptors or IL‐1 receptors, IL‐1 receptor associated kinases (IRAKs) are activated to initiate cascades of events that are critical in innate immune signaling pathways. The Pellino family of RING E3 ubiquitin ligases is an important regulator of IRAKs. All 3 human Pellino isoforms bind to and polyubiquitinate activated, phosphorylated IRAK1. We have determined the X‐ray crystal structure of the Pellino2 N‐terminal substrate‐binding region, which contains a previously unidentified non‐canonical example of a forkhead‐associated (FHA) domain. FHA domains are phosphoThr (pT)‐binding modules that mediate protein‐protein interactions in a variety of cell‐signaling pathways. We have shown that the canonical pT‐binding region of Pellino mediates interaction with pTs on IRAK1. We expect specificity is defined by non‐canonical features of the Pellino FHA domain and explore this with mutational analysis. We also use IRAK1 truncation variants to define a minimal region of phosphorylated IRAK1 that is sufficient for Pellino binding. This research is supported by a Career Award in the Biomedical Sciences from the Burroughs Wellcome Fund and by NIH Training Program (T32‐GM08275).

Compression garments (CGs) such as bandages and stockings are critical in the management of a wide range of lower extremity conditions, particularly venous leg ulcers (VLUs). In order for CGs to deliver therapeutic benefit, the interface pressure between the fabric and the skin much typically reach at least 30 mmHg. However, this threshold value is highly sensitive to the type of CG material used, correct placement by a trained healthcare provider, body position, and leg volume changes. Currently, PicoPressÒ (Microlabitalia, Padua, Italy) is the gold-standard tool to measure interface pressure. Unfortunately, the system is expensive, and requires an air-bladder wired to a large base unit, limiting the system to only point measurements. Using advanced materials science techniques, we report the development of a soft, wearable, flexible and wireless pressure sensor that consists of a gold thin wire (50 nm) integrated on a 3D polyimide structure and elastomer (Ecoflex-30, 2.4 mm in diameter, 1 mm in thickness). Once packaged, this low cost sensor is 15 mm x 45 mm can be comfortably placed between CGs and the skin without the risk of skin injury. Data is transmitted wirelessly to any standard smartphone. Testing in healthy normal subjects, the 3D sensor measured an interface pressure within 10% agreement with the PicoPressÒ across the full range of pressures (0 to 120 mmHg) by detecting the deformation of the elastomer (R 2 >0.98). A wearable interface pressure sensor will enable more effective compression therapy with CGs at the point of application and in the home setting. Future directions include evaluating the performance and tolerability of the device on patients with an active history of VLUs.